T_H1, T_H2, and T_H17 cells instruct monocytes to differentiate into specialized dendritic cell subsets

DCs are phenotypically and functionally divergent antigen presenting cells that originate from bone marrow precursors such as monocytes.^1-6  Encompassing a broad anatomical distribution, DCs continuously sample the microenvironment in search of danger signals recognized by a wide spectrum of pattern recognition receptors (PRRs).^1,2,6  Depending on the cytokine milieu and the pathogen encountered, DCs differentially up-regulate costimulatory molecules and secrete a variety of cytokines that dictate the nature of the T cell response.^7-10  DC exposure to particular intracellular pathogens or their products, including lipopolysaccharide (LPS) and double-stranded RNA, leads to DC-driven T_H1 differentiation primarily through IL-12 secretion.¹¹  Conversely, DCs mediate T_H2 differentiation in response to extracellular parasites, such as S mansoni, which likely results from diminished IL-12 secretion and increased OX40L expression.^7,8  Several reports have implicated DC-derived IL-1β, IL-6, IL-23, and TGF-β in the polarization of T_H17 cells,^9,12-17  which mediate defense against extracellular bacteria and various fungal infections.¹⁵ 

Psoriasis and atopic dermatitis are common inflammatory skin diseases characterized by the rapid accumulation of T_H cells and DCs.^18-22  While both diseases share several pathologic features,^18,22  the T_H cells implicated in the pathogenesis of each disease are distinct.^19,20,23  Infiltration of T_H1 and T_H17 cells is commonly observed in psoriatic lesions,¹⁹  which is accompanied by elevated levels of the T_H1 cytokine, IFN-γ, and the T_H17 cytokines, IL-17A and IL-22.¹⁹  Conversely, acute atopic dermatitis lesions contain elevated levels of T_H2 cells and their associated effector cytokines, IL-4, IL-5, and IL-13.^20,23  Not surprisingly, DCs that accumulate in each of these diseases exhibit markedly different phenotypes.^21,24,25  DCs present in psoriatic lesions express IL-12p40, IL-23, and TNF-α^25-27  while DCs present in atopic dermatitis lesions express low or undetectable levels of these cytokines.²⁵  DCs from each of these diseases also differentially express cell surface molecules such as DC-SIGN.²¹  However, the cell types and/or factors that influence the generation of these DC subsets remain elusive.

Given that memory T_H cells and monocytes rapidly infiltrate inflamed tissues²  and that activated T_H cells secrete high levels of GM-CSF,²⁸  a cytokine that mediates DC differentiation from monocytes,^29,30  we hypothesized that T_H cells instruct monocytes to differentiate into DCs. Our data demonstrate that not only do T_H cells direct monocytes to differentiate into DCs, but that each T_H cell subset drives the formation of phenotypically and functionally distinct DC subsets that resemble DCs previously identified in atopic dermatitis and psoriatic lesions.^21,24,25  These data support the hypothesis that memory T_H cells influence the nature of the secondary immune response by regulating DC formation and function.

Enriched CD4⁺ T cells and CD14⁺ monocytes were isolated from PBMCs obtained from healthy blood donors (Stanford Blood Center) by density gradient centrifugation using a custom RosetteSep cocktail (Stem Cell Technologies) containing mAbs against CD235a, CD8, CD19, CD20, CD56, CD66b and TCRγδ. CD14⁺ monocytes were isolated to > 97% purity using anti-CD14 conjugated microbeads (Miltenyi Biotec), and memory T_H cells were purified from the CD14⁻ fraction using a Memory CD4⁺ T cell Isolation Kit (Miltenyi Biotec). To sort unfractionated T_H cells, enriched memory T_H cells were stained with fluorescently labeled mAbs against CD14, CD16, CD19, CD25, CD36, CD56, CD123, CD235a, CD45RA, CD45RO (eBioscience), CD4 and CD8 (BD Bioscience) along with propidium iodide (PI; Invitrogen). T_H cells were defined as PI⁻CD4⁺CD45RA⁻CD45RO⁺CD25^−/lo. To sort for each T_H subset, enriched memory T cells were stained with fluorescently labeled mAbs against CD45RA, CD4 (Invitrogen), CD25, CD127 (Biolegend), CD161, CCR4, CCR6 and CRTH2 (BD Biosciences) along with PI. T_H1 cells were defined as PI⁻CD4⁺CD45RA⁻CD25^−/loCRTH2⁻CD161⁻CCR4⁻CCR6⁻; T_H2 cells were defined as PI⁻CD4⁺CD45RA⁻CD25^−/loCRTH2⁺; T_H17 cells were defined as PI⁻CD4⁺CD45RA⁻CD25^−/loCRTH2⁻CD161⁺CCR4⁺CCR6⁺.

Monocytes (1 × 10⁶) were cultured with or without 1 × 10⁵ autologous or allogeneic T_H, T_H1, T_H2 or T_H17 cells in 12-well plates (Corning) containing IMDM medium (Gibco) supplemented with 10% FCS, 2% human AB serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 2mM l-glutamine, sodium pyruvate, nonessential amino acids, 50μM 2-ME and, where indicated, 20 ng/mL IL-2 (Peprotech). GM-CSF and IL-4 derived DC (DC_GM) were generated as described previously.³¹  For imaging studies, cells were evaluated at day 6 by Leica DMIRB microscopy with a 40×/0.55 Hoffman Modulation Contrast objective and images were acquired using a digital ORCA-ER camera and Openlab acquisition software. Otherwise, cells were processed on day 6 with 5mM EDTA and subsequently stained with DAPI (Invitrogen), fluorescently labeled isotype control mAbs, or mAbs against CD11c, CD40, CD123 (Biolegend), CD14 (eBioscience), CD3, CD80, CD83, CD86, CD205, CD209 (BD Bioscience) or CD303 (Miltenyi Biotec). Cells were also stimulated at day 6 with 2 μg/mL LPS, 5 μg/mL Pam3CSK4, 10 μg/mL CpG, 10 μg/mL zymosan, 25 μg/mL poly(I:C), 1 μg/mL flagellin, 5 μg/mL imiquimod, 1 × 10⁸ cells/mL heat killed Listeria monocytogenes (HKLM), 1 μg/mL 3M-002 or 2 μg/mL N-glycol MDP for 36 hours and stained with the aforementioned mAbs against DC surface molecules or incubated with 100 μg/mL DQ-OVA (Invitrogen) on ice or at 37°C for an additional 30 minutes. Cells were analyzed on a BD LSRII flow cytometer, and FACS plots and MFIs were generated by FlowJo (Treestar). T cells were excluded according to their FSC:SSC profiles and CD3 expression. Cell-free supernatants were collected from LPS stimulated cultures for measurement of IL-1β, IL-6, IL-10, IL-12p70, IL-23p19, and TNF-α by ELISA. For neutralization studies, isotype control mAbs or mAbs against GM-CSF (Biolegend), CD119 (BD Bioscience), IL-17R, IL-21R (R&D Systems), IL-4R, IL-13, IFN-γ, TNF-α, or CD40L (eBioscience) were added at the initiation of culture at 10 μg/mL. Transwell experiments were performed using 24-well 0.4μM transwell inserts (Corning) with T_H cell and monocytes placed in the upper insert and monocytes below the insert or with T_H cells placed in the upper insert and monocytes in the lower insert.

Autologous T_H cells were magnetically depleted from LPS-stimulated monocytes, DC_Th and DC_GM using biotinylated anti-CD3 and anti-TCRα/β mAbs followed by anti-biotin microbeads (Miltenyi Biotec). Cells were irradiated (3000 rad) and cultured for 6 days at a 1:5 ratio with allogeneic naive CD4⁺CD45RA⁺CD45RO⁻CD25⁻ T cells isolated from PBMCs with a Naive CD4⁺ T Cell Isolation Kit II (Miltenyi Biotec). Cultures were pulsed with 1 μCi/well of ³H-thymidine for the last 20 hours and harvested by Harvester 400 (Tomtec), and radioactivity was measured by a 1450 MicroBeta counter (LKB Wallac). For DC_Th subsets, monocytes and T_H cells were cultured as described in “Monocyte/T-cell cocultures,” and after 6 days, T cell–depleted DC_Th (> 95% by magnetic depletion; > 99% by FACS purification) were stimulated with 2 μg/mL LPS for 36 hours and subsequently cultured at a 1:1 ratio with allogeneic naive CD4⁺CD45RA⁺CD45RO⁻CD25⁻ T cells. After 6 days, supernatants were assayed for IFN-γ, IL-5, IL-13, and IL-17A by ELISA.

Sections were deparaffinized in 3 changes of xylene followed by graded alcohols. Antigen retrieval was performed using pH 6.0 Diva universal retrieval solution (Biocare Medical) inside a pressure cooker (Biocare Medical). Endogenous peroxidase activity was quenched using 3% H₂0₂ (Lab Vision). Sections were blocked with serum-free protein blocker (Dako). For immunohistochemical stains in which primary antibodies were raised in different species, sections were incubated with a cocktail of mouse and rabbit primary antibodies inside a moist chamber followed by detection using a MACH-2 detection kit (Biocare Medical). Color development was performed with freshly prepared 3,3′-diaminobenzidine tetrahydrochloride (DAB) solution (Vector Labs) and Ferangi Blue solution (Biocare Medical) before mounting with Faramount Mounting Medium (Dako).

For stains in which the primary antibodies were raised in the same species, the protocol was carried out sequentially. The first antibody was detected with either an alkaline phosphatase or HRP-conjugated polymer kit (Biocare Medical) and developed in fresh DAB solution. Before the addition of the next primary antibody, the first reaction complex was treated with denaturing solution (Biocare Medical).

The primary antibodies used were: mouse monoclonal anti-CD4 (1:10, Vector), rabbit polyclonal anti-CD14 (1:200; Atlas), mouse monoclonal anti-GATA-3 (1:100; Santa Cruz Biotechnology), rabbit monoclonal anti–T-bet (1:100; Santa Cruz Biotechnology) and rabbit polyclonal anti-RORγ (1:40; Novus Biologicals).

Sections were evaluated by an Olympus BX51 microscope with a 40×/0.95 objective and images were acquired using a CRI Nuance VIS Flex camera and CRI Nuance acquisition software. Images were subsequently imported into ImageJ (National Institutes of Health).

Antibody pairs for IL-1β, IL-4, IL-5, IL-10, IL-17A, IL-23p19 (eBioscience), IL-13, and GM-CSF (BD Bioscience) were used for ELISA analysis. BD OptEIA kits were used for IL-6, IL-12p70, and TNF-α measurements. Samples were run in duplicate, and data and statistics were analyzed with GraphPad Prism. A 2-tailed Wilcoxon rank-sum test was used to analyze all pairwise comparisons between conditions. Two-tailed Wilcoxon rank-sum tests were also used to analyze the flow cytometry data.

Infiltration of tissues with T_H cells is a distinguishing feature of a variety of inflammatory diseases, including atopic dermatitis and psoriasis.^19,20,22  To investigate the possible interplay between monocytes and T_H cells in situ, skin biopsies from patients with atopic dermatitis and psoriasis were stained by immunohistochemistry with anti-CD4 and anti-CD14 antibodies. In both diseases, CD4⁺ and CD14⁺ cells were found in close apposition, suggesting that T_H cells interact with monocytes in inflamed skin (Figure 1A).

After infiltrating inflamed tissues, monocytes can differentiate into DCs.^3-5  To determine whether T_H cells trigger monocyte differentiation, FACS purified memory CD4⁺CD45RO⁺CD45RA⁻CD25^−/lo T_H cells from healthy donors and autologous CD14⁺ monocytes were cocultured with IL-2 for 6 days. Monocytes cultured under these conditions (DC_Th) underwent morphologic changes similar to those of monocyte-derived DCs generated with GM-CSF and IL-4 (DC_GM; Figure 1B). Multicolor flow cytometry analysis indicated that DC_Th and DC_GM expressed similar levels of DC-associated molecules such as CD11c, CD80 and CD209 (DC-SIGN; Figure 1C). DC_GM expressed lower levels of CD14, CD40 and CD123 in comparison to DC_Th (Figure 1C). Both cell types expressed high levels of CD11c (Figure 1C) and low or undetectable levels of CD303 (BDCA-2; supplemental Figure 1A, available on the Blood Web site; see the Supplemental Materials link at the top of the online article), which is consistent with the phenotype of myeloid DCs.³²  Conversely, monocytes cultured in medium alone, IL-2 alone (Mono) or with T_H cells alone did not exhibit an altered morphology and uniformly expressed low levels of DC-associated molecules (Figure 1B-C and supplemental Figure 1A-B). Moreover, monocytes cocultured with T_H cells and nominal antigens, such as tetanus toxoid and influenza peptides, or monocytes cocultured with T_H cells and IL-15, differentiated into DCs with a phenotype similar to that described in Figure 1B and C, (supplemental Figure 1C,E and data not shown). Addition of IL-2, IL-15, or nominal antigens in this system was necessary for GM-CSF secretion by T_H cells (supplemental Figure 1D and data not shown). These data indicate that T_H cells instruct monocytes to form DCs under a variety of conditions.

On activation, DCs increase the expression of costimulatory molecules, exhibit reduced endocytic activity and transform into potent antigen presenting cells.⁶  To assess the functional capacity of DC_Th, cells were cultured in the presence or absence of LPS for 36 hours. Under these conditions, DC_Th up-regulated the expression of costimulatory molecules CD80 and CD86 to comparable or higher levels than DC_GM (Figure 1D). After LPS stimulation, DC_Th and DC_GM exhibited reduced endocytic activity as measured by DQ-OVA fluorescence (Figure 1E). As expected, cells that were cultured on ice with DQ-OVA failed to internalize antigen (Figure 1E). To assess the capacity of DC_Th to stimulate naive T cell proliferation, T cell–depleted DC_Th were irradiated and subsequently cultured with allogeneic naive CD4⁺CD45RA⁺CD45RO⁻CD25⁻ T cells in a mixed leukocyte reaction (MLR). Both LPS-stimulated and unstimulated DC_Th induced naive T cell proliferation to a similar extent as DC_GM (Figure 1F). Taken together, these data indicate that DC_Th and DC_GM are functionally similar.

To investigate whether cell contact is required for DC_Th differentiation, monocytes and autologous T_H cells were cocultured with IL-2 on opposite sides of a 0.4 μm transwell membrane that allowed for the diffusion of soluble molecules, but not cells. Monocytes cultured under these conditions remained CD14^hi and did not up-regulate CD40 or CD123 (Figure 2A-B). DC-SIGN also remained low when monocytes were separated from T_H cells by a transwell membrane (Figure 2A-B). These data indicate that direct cell-cell contact is required for DC_Th differentiation.

To identify soluble or cell surface molecules capable of influencing DC_Th differentiation, monocytes and autologous T_H cells were cocultured with IL-2 and isotype control or neutralizing mAb against GM-CSF, TNF-α or CD154 (CD40L). Analysis of cocultures on day 6 demonstrated that GM-CSF neutralization inhibited DC_Th differentiation based on reduced acquisition of DC-SIGN and CD123 (Figure 2C). As expected, GM-CSF neutralization did not affect CD14 levels.³⁰  Conversely, TNF-α neutralization inhibited CD14 down-regulation and CD40 up-regulation (Figure 2C). Neutralization of GM-CSF or, to a lesser extent, TNF-α also prevented the acquisition of DC-like morphology (data not shown). These data suggest that GM-CSF and TNF-α play synergistic roles in DC_Th differentiation. Blocking CD40-CD40L interactions did not appear to alter DC differentiation (Figure 2C), which was surprising given the high levels of CD40 expression on DC_Th (Figure 1C).

To investigate the potential interactions between T_H subsets and monocytes in atopic dermatitis and psoriatic lesions, T_H1, T_H2, and T_H17 cells were identified according to the expression of their master regulatory transcription factors T-bet, GATA-3 and RORγ, respectively.^33-35  In both diseases, the majority of T-bet⁺, GATA-3⁺ or RORγ⁺ cells were CD4⁺, which is consistent with their identity as T_H cells (Figure 3A). As expected, infiltrating GATA-3⁺ cells in atopic dermatitis lesions were present at higher levels than T-bet⁺ and RORγ⁺ cells (supplemental Figure 2A and data not shown). Numerous CD14⁺ monocytes were also present in the infiltrate and were frequently juxtaposed to and/or in multipoint contact with GATA-3⁺ cells (Figure 3B). In contrast, most infiltrating CD4⁺ cells in psoriatic skin lesions were T-bet⁺ or RORγ⁺ (supplemental Figure 2A), and many of these cells were in close proximity with CD14⁺ cells (Figure 3B). Activated DCs, identified by CD208 (DC-LAMP) expression, were also found in close vicinity and/or multipoint contact with T_H cells in atopic dermatitis and psoriasis (supplemental Figure 2B). Given that DCs that arise in the presence of T_H1/T_H17 cells in psoriasis are phenotypically and functional divergent from DCs formed in the presence of T_H2 cells in atopic dermatitis,^21,24,25  we investigated the impact of different T_H cell subsets on DC_Th differentiation.

To determine whether T_H cell subsets are sufficient to generate DC diversity, we FACS purified T_H1, T_H2, and T_H17 cells from healthy donors^36-38  (supplemental Figure 2C-D) and subsequently cocultured each subset with allogeneic monocytes to simulate an inflammatory setting. Similar to monocytes cocultured with unfractionated T_H cells, monocytes cocultured with each T_H subset differentiated into DCs that resembled the DCs described in Figure 1C (supplemental Figure 2E). More importantly, each T_H subset elicited the formation of phenotypically distinct DC_Th subsets. DC_Th2 routinely expressed higher levels of DC-SIGN and CD275 (ICOSL; Figure 3C), which is consistent with reports indicating that T_H2-associated cytokines increase DC-SIGN and CD275 expression.^39-41  Conversely, DC_Th1 expressed elevated levels of CD86 and the inhibitory receptor, CD274 (PDL-1; B7-H1), but reduced levels of CD80 (Figure 3C).

To examine the cytokine secretion profile of each DC_Th subset, cell-free supernatants from LPS-stimulated cultures were analyzed by ELISA. Similar to previously purified DCs from psoriatic lesions,^25,27  DC_Th1 and DC_Th17 secreted high levels of IL-1β, IL-6, IL-23, and TNF-α (Figure 4A). DC_Th1 also secreted elevated levels of IL-12p70 (Figure 4A), a potent T_H1 polarizing cytokine that inhibits T_H2 and T_H17 differentiation.^42,43  Intracellular analysis of IL-1β, IL-12p40, and TNF-α confirmed these results (supplemental Figure 3A-B). Conversely, DC_Th2 more closely resembled DCs previously purified from atopic dermatitis lesions^21,25  and produced little or no TNF-α, IL-12p70, and IL-23 (Figure 4A). DC_Th2 also produced high levels of IL-10 (Figure 4A). Similar cytokine secretion profiles were observed with DC_Th generated with autologous T_H cells or by DC_Th stimulated with LPS in the absence of T_H cells (supplemental Figure 3C-D). Overall, these data indicate that cytokines secreted by DC_Th1 would be expected to promote a T_H1 response,^11,42  whereas the secretion of IL-1β, IL-6 and IL-23, but not IL-12p70, by DC_Th17 would be expected to promote a T_H17 response.^12-16  Similarly, the DC_Th2 cell surface and cytokine profile favors a T_H2 response.^7,8,42,43 

Given the marked differences in the cell surface and cytokine secretion profiles among the DC_Th subsets, we investigated whether each subset could differentially impact naive CD4⁺ T-cell polarization. Magnetically purified DC_Th subsets from autologous cocultures were stimulated with LPS for 36 hours and cultured in the presence or absence of allogeneic naive CD4⁺CD45RA⁺CD45RO⁻CD25⁻ T cells in an MLR. After 6 days, cell-free supernatants were assayed for the levels of IL-5, IL-13, IL-17A, and IFN-γ by ELISA. Notably, naive T cells cultured in the presence of DC_Th1 secreted elevated levels of the prototypic T_H1 cytokine, IFN-γ (Figure 4B). In contrast, DC_Th2 induced naive T cells to produce elevated levels of IL-5 and IL-13, indicative of a T_H2 response, while naive T cells cocultured with DC_Th17 secreted higher levels of IL-17A (Figure 4B). These data were confirmed by intracellular cytokine staining (supplemental Figure 4A). Because magnetic beads were used for DC_Th isolation, it was possible that residual T_H cells from the original cocultures could confound the cytokine data. However, similar results were obtained with FACS purified DC_Th subsets (> 99% purity; data not shown). Furthermore, the stated cytokines were largely undetectable when DC_Th subsets were cultured in the absence of naive T cells (supplemental Figure 4B) indicating that the contribution of residual T_H cells was negligible. These data indicate that each DC_Th subset promotes an immune response that mirrors the effector function of the T_H cell that initiated DC_Th differentiation.

To dissect the mechanisms governing the formation of distinct DC_Th subsets, monocytes and allogeneic T_H cells were cocultured in the presence of isotype control or T_H-specific neutralizing mAb. Analysis of cocultures on day 6 indicated that IFN-γ neutralization in DC_Th1 cocultures reduced the acquisition of a DC_Th1 phenotype as assessed by decreased CD86 and CD274 expression, but increased CD80 expression (Figure 5A). IFN-γ neutralization in DC_Th1 cocultures and IL-4R/IL-13 neutralization in DC_Th2 cocultures reduced the acquisition of a DC-like phenotype based on increased CD14 expression and decreased DC-SIGN expression (Figure 5A). Down-regulation of DC-SIGN after IL-4R/IL-13 neutralization is consistent with the high levels of DC-SIGN on DC_Th2 in comparison to the other DC_Th subsets (Figure 3C). Neutralization of IL-17R/IL-21R or IL-22 did not alter the surface phenotype of DC_Th17 according to any of the markers tested (Figure 5A and data not shown).

To determine which T_H-specific cytokines influence DC_Th cytokine secretion, DC_Th subsets were generated as in Figure 4 and stimulated with LPS in the presence of the appropriate isotype control or neutralizing mAb. After 36 hours, cell-free supernatants were analyzed by ELISA. The data indicate that IFN-γ neutralization reduced the acquisition of a DC_Th1 phenotype in favor of a DC_Th2 phenotype as assessed by increased IL-10 secretion and reduced secretion of IL-12p70, IL-23, and TNF-α (Figure 5B). Similarly, neutralization of IL-4R and IL-13 in DC_Th2 cocultures shifted the acquisition of a DC_Th2 phenotype toward a DC_Th1/DC_Th17 phenotype as assessed by increased IL-1β secretion (Figure 5B). Interestingly, neutralization of IL-17R/IL-21R or IL-22 did not alter the cytokine secretion profile of DC_Th17 (Figure 5B and data not shown) indicating that as of yet unidentified cytokines or cell surface molecules influence DC_Th17 formation and function.

To assess whether direct contact between T_H cells and monocytes was required for the formation of DC_Th subsets, we separated monocytes from monocyte-T_H cell cocultures with a 0.4-μm transwell membrane. Analysis of the cells on day 6 indicated that cell contact was necessary for the formation of each DC_Th subset based on the failure of monocytes separated from monocyte-T_H cell cocultures to down-regulate CD14 (Figure 5C). We also assessed the role of GM-CSF and TNF-α in the formation of DC_Th subsets by adding neutralizing mAb to monocyte-T_H cell cocultures. The data indicate that DC_Th1 and DC_Th17 formation required both GM-CSF and TNF-α, whereas DC_Th2 formation was independent of GM-CSF and partly dependent on TNF-α (Figure 5D). Thus, T_H specific cytokines and direct T_H cell-monocyte contact are required for the full acquisition of a DC phenotype.

As previously mentioned, DC responses are modulated according to the type of pathogen encountered.^7,9,10  To assess whether memory T_H cells or PRR engagement dominate DC_Th responsiveness, DC_Th cocultures were stimulated with a range of PRR agonists (TLR1-9 and NOD2) for 36 hours. Surprisingly, analysis of cell-free supernatants indicated that differential PRR engagement modulated the total amount of cytokine secreted, but did not alter the overall cytokine secretion profile. For example, DC_Th2 consistently secreted high amounts of IL-10 and relatively low amounts of IL-1β, TNF-α, and IL-23 regardless of PRR engagement (Figure 6). Instances where IL-10 secretion was higher in DC_Th2 such as HKLM or zymosan stimulation were also associated with concomitant increases in IL-1β, IL-6, IL-23, and TNF-α (Figure 6). Similarly, DC_Th1 and DC_Th17 typically secreted higher levels of IL-1β, IL-6, IL-23, and TNF-α in comparison to DC_Th2 (Figure 6). While some minor exceptions exist (ie DC_Th1, poly:IC), increases or decreases in the levels of IL-1β, IL-6, IL-23, and TNF-α were generally associated with concomitant increases or decreases in the other cytokines of this group (Figure 6). Taken together, these data indicate that not only do memory T_H cells elicit DC formation from monocytes, but that once formed, the DCs retain their functional phenotype regardless of subsequent TLR stimulation.

Several studies have demonstrated that monocytes infiltrate inflamed tissues and differentiate into CD11c⁺ DCs in vivo.^3-5  However, the mechanisms responsible for DC formation have remained elusive. The present study extends these findings by elucidating the mechanism whereby human T_H cells drive DC differentiation from monocytes. Our results demonstrate that T_H cells are juxtaposed with monocytes in situ and convert monocytes into DCs in a cell contact, GM-CSF and TNF-α dependent manner. This T_H-mediated DC differentiation pathway proceeds upon IL-2 stimulation, which is consistent with our data demonstrating that IL-2 induces GM-CSF secretion from T_H cells. This study also indicates that T_H cells convert antigen-loaded (tetanus toxoid or influenza antigens) monocytes into DCs in the absence of exogenous stimulation. These data indicate that T_H cells may participate in the innate response by initiating a positive feedback loop with monocytes and DCs that govern an intensified and polarized immune response.

One of the striking findings of this study is the capacity of each T_H subset to elicit the formation of specialized DC subsets. The data indicate that T_H1 cells instruct monocytes to form DCs that secrete IL-12 and express increased CD86 and CD274, whereas T_H17 cells elicit the formation of DCs that secrete IL-1β, IL-6 and IL-23, but not IL-12. The cytokines secreted by DC_Th1 and DC_Th17 are among the key cytokines used in vitro to generate T_H1 and T_H17 cells, respectively.^14,15  Interestingly, T_H2 cells drive the formation of T_H2-biased DCs that express elevated levels of IL-10, CD275 and DC-SIGN. While CD275 is known to promote IL-4 responses, IL-10 and DC-SIGN have been implicated in the inhibition of T_H1 and T_H17 responses, respectively.^17,44  Subsequent use of DC_Th1 and DC_Th17 as stimulators in an MLR induced responding CD4⁺ T cells to produce IFN-γ and IL-17A, respectively. While the identification of DC-derived soluble factors that influence T_H2 differentiation remains elusive,¹⁰  DC_Th2 nevertheless promoted IL-5 and IL-13 secretion from naive T cells. This implies that unidentified soluble factors or cell surface molecules such as OX40/OX40L, CD275 or DC-SIGN may be involved.^7,8,41  Gene expression profiling of each DC_Th subset may prove useful for identifying additional molecules involved in the formation and functions of DC subsets.

Our data indicate that distinct sets of T_H-derived molecules were responsible for DC_Th formation and polarization. Whereas all memory T_H subsets produced GM-CSF and TNF-α, the 2 cytokines required for generating DC morphology in our system, IFN-γ produced by T_H1 cells was required for the induction of DC_Th1 while IL-4 and IL-13 produced by T_H2 cells played requisite roles in the induction of DC_Th2. Interestingly, T_H17 cell-derived IL-17, IL-21, or IL-22 were not required for DC_Th17 formation. Moreover, monocytes or DC_GM cultured with LPS in the presence of T_H17-associated cytokines, IL-17A and/or IL-21, did not secrete different cytokines compared with cells cultured with LPS alone (M.N.A., M.T.W., and E.G.E., unpublished data, August 2010). These data suggest that yet to be identified soluble and/or cell surface molecules may be involved in DC_Th17 differentiation.

Taken together, our results indicate that T_H cells positively regulate the secondary immune response by instructing monocytes to differentiate into DCs, which in turn trigger naive CD4⁺ T cells to acquire similar effector functions as the T_H cell subset that initiated DC differentiation. Such an amplification loop would be beneficial during memory responses, but deleterious if uncontrolled during inflammatory diseases. Psoriasis and atopic dermatitis are 2 diseases that likely involve divergent feedback loops, as evidenced by the accumulation of markedly different T_H cells and DC subsets in inflamed skin.^18-22  Psoriatic lesions contain primarily T_H1 and T_H17 cells,¹⁹  and possess CD11c⁺ myeloid DCs that strongly resemble the functional characteristics attributed to DC_Th1 and DC_Th17.^24-27 Most notably, CD11c⁺ DCs in psoriatic lesions express elevated levels of TNF-α, IL-12p40, and IL-23^25,27  and drive the formation of T_H1 and T_H17 cells ex vivo.²⁴  Conversely, atopic dermatitis lesions contain large numbers of T_H2 cells^20,22,23  and CD11c⁺ DCs that share several characteristics with DC_Th2.^21,25  Importantly, DCs from atopic dermatitis lesions express low or undetectable levels of TNF-α, IL-12p40, and IL-23^21,25  and are believed to mediate T_H2 differentiation during the acute phase of atopic dermatitis.²²  DCs in atopic dermatitis also express higher levels of DC-SIGN compared with DCs from psoriatic lesions,²¹  which is in accordance with increased DC-SIGN expression observed on DC_Th2. These data indicate that CD11c⁺ DCs present in inflamed skin from classic T_H-associated diseases are phenotypically similar to those described in this report.

The numerical predominance of T_H cells in lesions from psoriasis and atopic dermatitis patients indicates that these cells may be principally responsible for shaping the local DC repertoire. However, this does not exclude the possibility that monocytes also receive signals from other cell types. Previous studies have shown that NK, CD8⁺ and NK T cells can also initiate DC differentiation.^31,45,46  Similar to T_H cells, NK cells colocalize with monocytes in inflamed tissue and direct DC differentiation in a GM-CSF and cell contact dependent manner.⁴⁶  Comparison of the relative contributions of each of these cell types to inflammatory responses merits further investigation.

The memory immune response is characterized by rapid responses to foreign agents encountered 2 or more times throughout the lifetime of the host. While this response is partially mediated by affinity maturation⁴⁷  and differential activation requirements,⁴⁸  our data suggest that memory T_H cells may also contribute to the speed and potency of the secondary response by eliciting the formation of specialized DC subsets. Interestingly, stimulation of these DC subsets with a panel of PRR agonists which were previously demonstrated to modulate DC cytokine secretion,^7,9,10  affected total cytokine production, but failed to alter the relative ratios of individual cytokines produced by each DC_Th subset. These data indicate that, once formed, the phenotype of each DC_Th subset is stable and that signals from memory T_H cells outweigh subsequent signals from PRR engagement.

While our study elucidates a novel mechanism by which T_H cells can amplify the immune response, resolution of this response is essential to prevent ongoing inflammation and autoimmunity. Elimination of the initial immune stimulus, such as a pathogen or allergen, would presumably facilitate resolution. In addition, preliminary data from our laboratory indicate that regulatory T cells can instruct monocytes to differentiate into tolerogenic DCs, thereby providing another mechanistic brake on inflammation. It is also conceivable that T_H cells initiate a negative feedback loop after multiple rounds of activation.⁴⁹  This is supported by a recent report indicating that irradiated T_H1 and T_H2 clones prompt the formation of DCs that subsequently induce naive T cells to secrete IL-10 and IFN-γ, respectively.⁵⁰  Thus, whereas T_H cells may mediate an amplification pathway during the initiation of a secondary immune response, a variety of mechanisms likely exist to limit the intensity and duration of such responses.

This study identifies an important mechanism responsible for generating DC diversity that also provides a critical link between innate and adaptive immunity. The system we used for generating DC subsets with differential T_H polarizing capacity has facilitated the elucidation of cell surface and soluble factors governing the biology of T_H-promoting DC subsets. These and additional molecules expressed by DC subsets should prove useful for detecting and quantifying each subset in situ and enable the design of novel therapeutic strategies for T_H-associated inflammatory diseases.

The authors would like to extend our special thanks to Toru Iwahori and Kartoosh Heydari for their expert assistance in flow cytometry. They thank Donna Jones for secretarial assistance and Claudia Benike, Oliver Crespo, Luis Zuniga, Stephanie Fung and Christina Swanson for careful review of the manuscript.

This work was supported by the following funding sources: National Heart, Lung, and Blood Institute (HL075462), 5 U19 AI050864, 1 U19 AI082719, 1 U19 AI090019, and gifts from the Floren Family Foundation and the Ben May Trust. M.N.A. was funded by National Institutes of Health Fellowship (NRSA) F31 CA142272. M.T.W. was funded by a Mason Case Fellowship.

National Institutes of Health

Contribution: M.N.A., M.T.W., and A.L.Z. conceived and executed the studies; M.M.S. and M.G.D. assisted with experimental design and provided technical guidance; D.W. and J.G. performed the IHC studies; T.H.K., D.M.G., and L.T. assisted in the execution of select studies; K.C.D. and J.K. provided psoriasis and atopic dermatitis tissues; P.J.U. supervised select studies. K.S. and E.G.E. conceived and supervised the studies; M.N.A., M.T.W., A.L.Z., M.M.S., D.W., M.G.D., P.J.U., K.S., and E.G.E. analyzed and/or interpreted the data; and M.N.A., M.T.W., and E.G.E. wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

The current affiliation for K.S. is Novo Nordisk Inc.

Correspondence: Dr Edgar Engleman, Stanford University School of Medicine, 3373 Hillview Ave, Palo Alto, CA 94304-1204; e-mail: edengleman@stanford.edu.