T Cell Immunity +CD4 Cell-Restricted MHC Class II Controls Th1

CD8α+ and CD11b+ Dendritic Cell-Restricted MHC Class II Controls Th1 CD4+ T Cell Immunity

This information is current as Maria P. Lemos, Lian Fan, David Lo and Terri M. Laufer of September 25, 2021. J Immunol 2003; 171:5077-5084; ; doi: 10.4049/jimmunol.171.10.5077 http://www.jimmunol.org/content/171/10/5077 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

CD8␣؉ and CD11b؉ Dendritic Cell-Restricted MHC Class II Controls Th1 CD4؉ T Cell Immunity1

Maria P. Lemos,* Lian Fan,2† David Lo,3† and Terri M. Laufer4*

The activation, proliferation, differentiation, and trafficking of CD4 T cells is central to the development of type I immune responses. MHC class II (MHCII)-bearing dendritic cells (DCs) initiate CD4؉ T cell priming, but the relative contributions of other MHCII؉ APCs to the complete Th1 immune response is less clear. To address this question, we examined Th1 immunity b in a mouse model in which I-A␤ expression was targeted specifically to the DCs of I-A␤b؊/؊ mice. MHCII expression is reconstituted in CD11b؉ and CD8␣؉ DCs, but other DC subtypes, macrophages, B cells, and parenchymal cells lack of expression b of the I-A␤ chain. Presentation of both peptide and protein Ags by these DC subsets is sufficient for Th1 differentiation of -Ag-specific CD4؉ T cells in vivo. Thus, Ag-specific CD4؉ T cells are primed to produce Th1 cytokines IL-2 and IFN-␥. Addi ؉ tionally, proliferation, migration out of lymphoid organs, and the number of effector CD4 T cells are appropriately regulated. Downloaded from However, class II-negative B cells cannot receive help and Ag-specific IgG is not produced, confirming the critical MHCII requirement at this stage. These findings indicate that DCs are not only key initiators of the primary response, but provide all of ,the necessary cognate interactions to control CD4؉ T cell fate during the primary immune response. The Journal of Immunology 2003, 171: 5077–5084.

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ajor histocompatibility complex class II (MHCII) B220 Ly6G/C plasmacytoid DC (pDCs). These DC subsets http://www.jimmunol.org/ molecules are required for the development of CD4ϩ have all been shown to regulate Th1 priming, but whether they T cells in the thymus and function to present peptide play redundant or specific roles in T cell immune responses is M ϩ Ags to CD4 T cells in the periphery (1, 2). Constitutive expres- unknown (7Ð9). Thus, it is unclear whether cognate interactions sion of MHCII molecules at peripheral sites is limited to profes- with all DC types are necessary for normal Th1 immune responses, sional APCs such as macrophages, dendritic cells (DCs), and B or whether individual cell types direct polarization of Th1 cells in cells. MHCII expression can also be induced on endothelial and a noncognate manner. parenchymal tissues by inflammatory stimuli such as IFN-␥ (3). Mature DCs have a very short life span (10); therefore, T cell Although peripheral MHCII expression on DCs mediates the sur- ϩ interactions with other APCs may contribute to complete primary vival of CD4 T cells (4), the specific role of individual MHCII- by guest on September 25, 2021 ϩ immune responses (11). Thus, B cells may contribute to the full positive APCs during CD4 T immune responses has not been expansion of Ag-specific CD4 T cells to drive Th2 differentiation, fully elucidated. ϩ but whether this requires Ag presentation is controversial (12Ð14). Priming naive CD4 T cells is largely the function of DCs, However, the effects of B cell Ag presentation on Th1 CD4ϩ dif- which present Ag in the context of high levels of MHC, costimu- ferentiation are unknown. latory molecules, activating cytokines, and T cell-attracting che- The differentiation and function of Th1 effector cells relies on mokines (5, 6). DCs can be distinguished from other class II-pos- TCR-MHCII interactions, proliferation, and the cytokine milieu. itive APCs by the expression of the integrin pair CD11c/CD18 (6). ϩ The strength of the MHCII-TCR signal plays an important role in CD11c DCs can be further divided into multiple subsets based on Th1 polarization (8, 15); therefore, the lack of MHCII from certain surface markers, anatomic localization, and cytokine production (5). Chief among these subsets are: CD11cneg-low epidermal Lang- APCs could affect the Th1/Th2 balance. MHCII-peptide-TCR in- erhans cells (LCs), CD11bϩ DCs, CD8␣ϩ DCs, and CD11clow teractions facilitate production of IL-12, which drives Th1 polarization, by macrophages and DCs (5, 16Ð18); thus, both APCs might be important in the differentiation and function of Th1 ef- *Department of Medicine, University of Pennsylvania, Philadelphia PA 19104; †The Scripps Research Institute, La Jolla, CA 92037 fectors. TCR-MHCII interactions also may contribute to the re- ϩ Received for publication June 17, 2003. Accepted for publication September cruitment and accumulation of CD4 T cells into areas of inflam- 15, 2003. mation (19), although recent evidence has suggested this event ϩ The costs of publication of this article were defrayed in part by the payment of page does not require Ag (20). Therefore, local MHCII macrophages charges. This article must therefore be hereby marked advertisement in accordance and parenchymal cells might be required for accumulation of ef- with 18 U.S.C. Section 1734 solely to indicate this fact. fector cells at infected sites. 1 This work was supported by a Grant-in-Aid from the Pennsylvania/Delaware affil- iate of the American Heart Association (to T.M.L.). Maturation signals to DCs and Ag dose have been reported to 2 Current address: Pharmacia, 4901 Searle Parkway, Stokie, IL, 60077. play a crucial role in Th differentiation (5, 8, 15, 21). Studies addressing the role of DCs in CD4ϩ T cell differentiation have 3 Current address: Digital Gene Technologies, 11149 North Torrey Pines Road, La Jolla, CA 92037. used DCs that have been matured and loaded with Ag in vitro and 4 Address correspondence and reprint requests to Dr. Terri M. Laufer, Department of then transferred i.v. or s.c. into hosts. We chose to evaluate the Medicine, University of Pennsylvania, 753 BRB II/III 421 Curie Boulevard, Phila- sufficiency of MHCII Ag presentation by DCs, allowing Ag up- delphia, PA 19104. E-mail address: [email protected] take, MHCII peptide loading, DC maturation, and migration to 5 Abbreviations used in this paper: MHCII, MHC class II; DC, dendritic cell; pDC, plasmacytoid DC; LC, Langerhans cell; BMDC, bone marrow-derived DC; MFI, occur in vivo. To do so, we used the CD11c promoter to target mean fluorescence intensity; LN, lymph node. MHCII expression exclusively to the DCs of class II-deficient

ϩ ϩ mice. We demonstrate that CD8␣ and CD11b DCs are suffi- (Sigma-Aldrich). Macrophage analysis excluded CD11chighCD86high DCs. cient for the expansion, differentiation, migration, and contraction Intracellular MHCII was performed on 1% paraformaldehyde-fixed sam- ϩ ples that were permeabilized with PBS/2% BSA/0.02% saponin. of Ag-specific Th1 CD4 T cells during the primary response. ϩ For intracellular cytokine staining, CD45.1 cells were purified using These findings indicate that Ag presentation by DCs not only con- CD45.1 biotin (BD PharMingen) and anti-biotin microbeads (Miltenyi trols priming events, but regulates the fate of Th1 effector CD4 T Biotec) on paramagnetic columns. Cells were treated with 50 ng/ml PMA cells throughout the response. and 500 ng/ml Ionomycin in the presence of 2 nM monensin for5hat37¡C. OTII cells (V␣2V␤5) were surface stained for V␤5, CD4, and CD45.1, followed by fixation and permeabilization as described above. Intracellular Materials and Methods cytokine staining was performed using anti-IL-2-allophycocyanin, IL-4- Transgenic mice allophycocyanin, and IFN-␥-allophycocyanin (BD PharMingen). The CD11c promoter (4) was obtained from Dr. I. Mellman (Yale Uni- b B cell and macrophage stimulation versity, New Haven, CT). The A␤ cDNA (22) (gift from Dr. R. Germain, National Institutes of Health, Bethesda, MD) was inserted into the EcoRI To obtain activated B cells, splenocytes were cultured for 48 h in DMEM b cloning site in the CD11c cassette (4). The linearized CD11c/A␤ construct with 10% FBS, 50 ␮g/ml LPS from Salmonella typhimurium (Sigma- ϫ was injected into (BALB/c C57BL/6)F1 eggs. Three different founder Aldrich), and 10 ng/ml recombinant mouse IL- 4 (PeproTech). Peritoneal lines were generated and backcrossed to C57BL/6 mice for 6Ð10 macrophages were harvested in 10 ml of 10% FBS in RPMI 1640 and generations. cultured with 100 U/ml IFN-␥ (PeproTech) for 72 h. C57BL/6, C57BL/6J-Tcratm1Mom (TCR␣Ϫ/Ϫ) (23), and B6.SJL-Ptpcrca Pep3b/BoyJ (CD45.1) congenics were obtained from The Jackson Labo- Bone marrow-derived DCs (BMDC) ratory (Bar Harbor, ME). I-A bϪ/Ϫ mice (2) were bred in our colony (22 ␤ BMDCs were generated as previously described (28) using recombinant generations backcrossed to C57BL/6 background). TEa mice (24), a gift mouse GM-CSF (PeproTech) or supernatants of B78H1/GM-CSF cell line Downloaded from from Dr. M. Jenkins (University of Minnesota, Minneapolis, MN), and cultures, a kind gift from Dr. H. Levitzky (Johns Hopkins University, Bal- OTII mice (25), a gift from Dr. C. Surh (The Scripps Institute, La Jolla, timore, MD) in the BMDC cultures for 8 days. For BMDC maturation, 1 CA), were bred to CD45.1 congenics. Mice 6Ð10 wk of age were used in ϩ ϩ ␮g/ml LPS was added 18 h before analysis of CD11c CD11b cells. Stu- all experiments. dent’s paired t test was used to determine statistical significance in the Immunohistochemistry maturation markers and MHCII levels. For peptide-pulsed BMDCs, on day 6, OVA peptide 323Ð339 (Univer- Cryosections of spleen and thymus (5 ␮m) were fixed in cold acetone, sity of Pennsylvania Cancer Center Peptide Synthesis Core) was added to ␮ http://www.jimmunol.org/ washed in PBS and 0.3% H2O2, and blocked with goat serum and avidin- the cultures in concentrations ranging from 0.01 to 3 g/ml; OVA protein biotin block (Vector Laboratories, Burlingame, CA). Samples were stained (Sigma-Aldrich) was added in concentrations ranging from 100 to 1 ␮g/ml. with primary Ab against I-Ab/d (M5/114) and CD3 (2C11; American Type Ag-pulsed BMDCs were washed three times and cultured with 1 ϫ 105 Culture Collection (ATCC), Manassas, VA). Adjacent sections were CFSE-labeled OTII CD4ϩ T cells for 4 days. stained with B220 (RA3; ATCC) and CD3 to define B and T cell zones. Primary Abs were detected with biotin-conjugated mouse anti-rat or anti- OTII CD4 T cell purification, priming, and analysis hamster IgG Fabs (Jackson ImmunoResearch, West Grove, PA) followed OTII CD4ϩ T cells were purified by positive selection of CD4ϩ cells on by the ABC Elite kit or Vectastain ABC Alkaline Phosphatase kit (Vector AUTOMACS columns (Miltenyi Biotec) and were 94Ð98% V␣2ϩ. CFSE Laboratories). HRP and alkaline phosphatase were developed using 3-ami- (Molecular Probes, Eugene, OR) labeling of OTII cells and quantitation of no-9-ethylcarbazole (Sigma-Aldrich, St. Louis, MO) or Vector Blue alka- CFSE dilutions were conducted according to previously published proto- line phosphatase kit (Vector Laboratories), respectively. cols (29, 30). Responder frequency is the proportion of the initial pool that by guest on September 25, 2021 DC isolation divides after stimulation (30). For in vivo experiments, three to five million OTII CD4ϩ T cells were Organs were dissected, placed in incomplete Iscove’s medium on ice, in- transferred i.v. into each recipient. Twenty-four hours later, mice were ␮ ␮ ␮ jected with 1 ml of 5 mg/ml collagenase type IV (Worthington Biochemi- immunized i.v. (31) with 100 g OVA323Ð339 and 75 g LPS, 300 g OVA cals, Lakewood, NJ) and 0.3% DNase I (Sigma-Aldrich), and incubated at protein (Sigma-Aldrich), and 75 ␮g LPS or 75 ␮g LPS (Sigma-Aldrich). room temperature for 30Ð45 min. Single-cell suspensions were washed To analyze OTII activation, lungs, spleens, and lymph nodes (LNs) were and resuspended in PBS with 2% BSA, 2 mM EDTA, polyclonal rat IgG analyzed by flow cytometry. Lungs from immunized mice were cultured in (Sigma-Aldrich), and CD11c MACS beads (Miltenyi Biotec, Auburn, CA). 10 ml of 0.5 mg/ml collagenase type IV (Worthington Biochemicals) and After staining, cells were washed and purified on paramagnetic AU- 0.1% DNase I (Sigma-Aldrich) and incubated at 37¡C for 30Ð45 min. TOMACS (Miltenyi Biotec) columns according to the manufacturer’s in- After digestion, lungs were dissociated into single-cell suspensions and ϩ structions. CD11cϩ cell purity ranged from 70 to 98% depending on the separated on Percoll gradients (32). OTII CD4 T cell recovery is based on ϩ ϩ ϩ organ. the number of CD45.1 CD4 V␤5 cells found in the preparations of LNs pDCs were analyzed by flow cytometry after positive selection of and spleen. Statistical tests for OTII recovery differences used Student’s CD45R/B220ϩ splenocytes on paramagnetic AUTOMACS columns paired t and Mann-Whitney U tests. (Miltenyi Biotec) using anti-CD45R-biotin (BD PharMingen, San Diego, CA) and anti-biotin microbeads (Miltenyi Biotec). LCs were purified from ELISA ear skin explants as described before (26, 27). In vitro culture of epidermal Anti-OVA IgG was measured by ELISAs as previously described (33) and dermal layers was conducted in complete RPMI 1640 with 100 ng/ml using diluted serum samples. IgG titers were determined using alkaline recombinant mouse GM-CSF (PeproTech, Rocky Hill, NJ) for 24 h. Mi- phosphatase-conjugated goat anti-mouse IgG (Southern Biotechnology As- grating LCs collected in the media were analyzed by flow cytometry using sociates, Birmingham, AL) and developed as previously described (53). anti-CD45 to identify the hemopoietic LCs. Flow cytometry Results CD8␣ϩ and CD11bϩ DCs are I-Ab positive in CD11c/A␤b mice Single-cell suspensions were blocked with Abs against FcRII/III (24G2; ATCC). The Abs used for staining were I-Ab-FITC (AF6), rIgG-FITC, To probe the sufficiency of MHCII-dependent Ag presentation me- CD11c-PE, human IgG2a-PE, CD8␣-PerCP, rIgG-PerCP, CD11b-allophy- diated by DCs, we have examined the phenotype of transgenic cocyanin, rIgG-allophycocyanin, CD45-Bio, CD45R-bio, CD86-FITC, mice in which I-Ab expression is restricted to DCs. Brocker (4) CD80-FITC, CD40-FITC, CD45-FITC, CD4-FITC, human IgG-FITC, mouse IgG-FITC, V␣2-FITC, I-Ab-PE, B220-PE, CD44-FITC, CD62L- previously reported that the murine CD11c promoter reconstitutes FITC, CD45RB-FITC, CD69-FITC, CD69-PE, CD8-FITC, CD4-PerCy, I-E␣ expression in 70% of thymic and splenic DCs in transgenic V␤5-PE, CD45.1-biotin, CD19-allophycocyanin, streptavidin-allophyco- C57BL/6 mice. We used this promoter to selectively re-express the cyanin, and streptavidin-FITC (BD PharMingen). Samples were analyzed b b I-A␤ chain in the I-A␤ Ϫ/Ϫ (MHCII-deficient) background. Two on a FACSCalibur (BD Biosciences, San Diego, CA) using CellQuest soft- 0 4 founder lines led to MHCII expression on 12 and 40% of DCs; a ware. All dot plots shown have a log axis of 10 Ð10 . Staining of macro- ϩ phages, DCs, and activated B cells was conducted in PBS with 2% BSA, third founder line had 90% of wild-type MHCII DCs and was 2 mM EDTA, 1 ␮g/ml mouse IgG (Sigma-Aldrich), and 1 ␮g/ml rat IgG used for all experiments. The Journal of Immunology 5079

b Transgene-positive mice were backcrossed to I-A␤ Ϫ/Ϫ mice fully restored MHC class II expression in this tissue (data not b (CD11c/A␤ mice) and analyzed for restoration of class II expres- shown). b b sion. Surface expression of I-A in CD11c/A␤ mice should occur We expected that DC subsets with low levels of CD11c, mainly b b in cells where transgenic I-A␤ expression overlaps with endoge- pDCs and LCs, would remain I-A negative and this was the case. nous I-A␣b expression. To quantitate the expression of I-Ab in- CD19ϪB220ϩCD11clow pDCs lacked signiﬁcant MHC class II ex- b b b/low duced by the transgene, we performed FACS analysis on DCs pression in CD11c/A␤ mice, whereas I-A␤ ϩ/Ϫ pDCs are I-A obtained from multiple organs. Equivalent numbers of DCs were (Fig. 1D). In agreement with a recent report that this promoter cassette b b b obtained from I-A␤ Ϫ/Ϫ, CD11c/A␤ , and I-A␤ ϩ/Ϫ mice, sup- did not drive expression in LCs (35), skin-resident Langerhans’ cells porting previous observations that MHC class II expression is not were also I-Ab negative and remained class II-negative following epi- required for normal DC development and localization (15, 34). In dermal irritation and migration into the draining LN (Fig. 1E and data b lymphoid organs, Ͼ90% of CD11c/A␤ DCs had surface MHCII not shown). Therefore, we suspect that the 10% of lymphoid organ b b expression greater than I-A␤ Ϫ/Ϫ-negative controls (Fig. 1A). DCs that remain class II-negative in CD11c/A␤ mice (Fig. 1A)in- b low Thus, the CD11c/A␤ transgene rescued MHCII expression on clude pDCs, LCs, and other CD11c subsets. most DCs within lymphoid organs. Immunohistochemistry was used to examine the localization of DCs can be phenotypically and functionally subdivided into the transgenic class II-positive DCs in lymphoid organs. In the multiple subsets with varying levels of CD11c. We analyzed thymus, anti-I-Ab staining was detected on scattered stellate cells MHCII expression within individual DC subsets. We ﬁrst subdi- in the medulla (Fig. 1F). These cells were also CD11c positive vided DCs isolated from spleens and LNs into CD8␣ϩ and (data not shown). Because CD11c-driven transgenes do not drive ϩ CD11b DCs. I-Ab reconstitution was incomplete in CD11blow expression in thymic epithelium, the pattern of MHCII expression Downloaded from high b b CD11c DCs from CD11c/A␤ mice (Fig. 1B); surface MHCII in CD11c/A␤ mice fails to drive positive selection of MHCII- b b expression was ϳ65% of the staining in I-A␤ ϩ/Ϫ DCs ( p ϭ restricted cells (data not shown), and CD11c/A␤ mice have no b ϩ ϩ b 0.009). In contrast, freshly isolated CD11c/A␤ CD8␣ DCs had more CD4 T cells than do I-A␤ Ϫ/Ϫ mice (36). wild-type levels of I-Ab (Fig. 1C). CD8␣ϩ DC are also the pre- Others have previously reported that splenic morphology is in- b dominant type of DCs in the thymus and the CD11c/A␤ transgene dependent of MHCII expression and the presence of T cells (15, http://www.jimmunol.org/ by guest on September 25, 2021

b b ϩ FIGURE 1. Characterization of MHCII expression in CD11c/A␤ mice. M1 refers to staining above I-A␤ Ϫ/Ϫ controls. A, Total CD11c DCs from spleen and LNs (n ϭ 8). B, CD8␣ϩ DCs (n ϭ 3). C, CD11bϩ DCs (n ϭ 5). D, CD19ϪB220ϩCD11clow pDCs (n ϭ 3). E, LCs (CD45ϩ cells migrating out of skin explants) (n ϭ 4). F, Immunohistochemistry of thymus sections stained with Abs against I-Ab (M5114). Representative photographs (n ϭ 3). C, Cortex; M, medulla. G, Immunohistochemistry of the spleen stained with Abs against I-Ab (red) and CD3 (blue). Representative photographs (n ϭ 3). T, T cell zones; B, B cell zones. H, intracellular MHCII on B220highCD19ϩ B cells (n ϭ 3). I, Surface MHCII on LPS-treated B cells (n ϭ 5). J, Surface MHCII on IFN-␥-treated peritoneal macrophages (n ϭ 4). 5080 DC Ag PRESENTATION CONTROLS CD4ϩ Th1 CELL RESPONSES

b b 34, 37). In agreement with these reports, CD11c/A␤ mice had (Fig. 1H) and intracellular I-A␤ expression (data not shown). Fi- b well-defined B cell and T cell zones (Fig. 1F). I-A␤ expression in nally, LPS-treated B cells could stimulate neither peptide-specific b ϩ CD11c/A␤ spleens localized to the T cell zones and scattered nor allogeneic CD4 T cells responses (data not shown), verifying areas of the red pulp and paralleled anti-CD11c staining of adja- that these APCs are functionally MHCII negative. b cent sections (data not shown). No I-A␤ staining of B cell areas To analyze MHCII expression in activated macrophages, peri- b b was detected in CD11c/A␤ spleens. Thus, CD11c/A␤ mice main- toneal macrophages were treated with IFN-␥ for 72 h and the tain normal splenic architecture and MHCIIϩ DCs are appropri- CD11bhighCD11clow macrophages were analyzed by flow cytom- ately localized. etry. Macrophages from all genotypes were activated with in- Finally, we analyzed resident tissue DC isolated from liver and creased surface levels of CD80 and CD86 (data not shown). Wild- b b b lungs. DCs purified from CD11c/A␤ lungs expressed equivalent type I-A␤ ϩ/Ϫ macrophages increased surface levels of I-A ;no b b b levels of I-A␤ when compared with I-A␤ ϩ/Ϫ lung DCs; in con- change was apparent in the level of I-A on activated macrophages b b trast, only a quarter of the DCs present in the liver of CD11c/A␤ from CD11c/A␤ mice, which remained MHCII negative (Fig. 1I). b mice were MHCII positive (data not shown). The MHCII expres- Therefore, CD11c/A␤ mice have MHCII expression restricted sion pattern correlated with the different DC subsets present in to DCs. these organs: lungs have mainly CD11bϪCD8␣ϩ DCs, whereas the liver contains a higher proportion of pDCs and b CD11bϩCD11cϩ DCs (38Ð40). In conclusion, CD11c-driven CD11c/A␤ DCs mature normally and present Ag in vitro b I-A␤ expression led to complete reconstitution of MHCII in The maturation phenotype (CD40, CD80, CD86) and DC yields of ϩ ϩ ␣ b b b Downloaded from CD8 DCs and slightly decreased I-A expression in CD11b DCs isolated ex vivo from I-A␤ ϩ/Ϫ mice and CD11c/A␤ mice DCs; pDCs and LCs remained MHCII negative. were comparable (data not shown), suggesting that DC maturation was not altered by the presence of the transgene. We verified that B cells and macrophages are class II negative in b b DC I-A expression was regulated appropriately during maturation CD11c/A␤ mice b in CD11c/A␤ mice by following the LPS-driven maturation of b To determine whether transgenic I-A␤ expression was limited to BMDCs. Immature BMDCs from all genotypes had comparable

DCs, B cells and macrophages were analyzed by flow cytometry. levels of CD80, CD86, and CD40 (Fig. 2A, black lines) and these http://www.jimmunol.org/ Resting B220ϩ splenic B cells and CD11bϩ peritoneal macro- increased equivalently following LPS-induced maturation (Fig. phages lacked surface expression of I-Ab heterodimers. Surface 2A, gray histograms). However, I-Ab expression (mean fluores- b b and intracellular staining with anti-A␤ -specific Abs (AF6) re- cence intensity (MFI)) in immature CD11c/A␤ BMDCs was two- b b b vealed no more protein in CD11c/A␤ B cells than in I-A␤ Ϫ/Ϫ B thirds that of immature I-A␤ ϩ/Ϫ BMDCs ( p ϭ 0.002). Immature b cells (Fig. 1G and data not shown). To ensure that this phenotype BMDCs from CD11c/A␤ mice resembled freshly isolated was stable with activation, splenic B cells were cultured with IL-4 CD11bϩ DCs and 5Ð10% of the population also lacked surface and LPS to generate activated B lymphoblasts and then reanalyzed. MHCII. Importantly, LPS-dependent maturation up-regulated I-Ab b B cells from all genotypes had equivalent up-regulation of CD69 in CD11c/A␤ BMDCs to levels similar to those of wild-type bϩ Ϫ ϭ Ϯ by guest on September 25, 2021 and CD86, indicating they were activated (data not shown). How- I-A␤ / BMDCs (ratio MFICD11c/A␤b/MFII-A␤bϩ/Ϫ 0.9 b b b ever, at least 99% of the CD11c/A␤ B cells lacked surface I-A 0.37). Thus, CD11c-driven I-A␤ expression is sufficient for wild-

b FIGURE 2. BMDCs from CD11c/A␤ mice have normal maturation and peptide presentation. A, Maturational markers of BMDCs (n ϭ 4). B and C, ϭ OTII CD4 T cell proliferation quantitated by CFSE responder frequency (n 3) in response to different concentrations of OVA323Ð339 peptide on BMDCs ␮ (B) or different numbers of BMDCs pulsed with 0.5 g/ml OVA323Ð339 (C). The Journal of Immunology 5081

b type I-A␤ surface expression of MHCII heterodimers in We first followed changes in phenotypic markers of activation mature DCs. following immunization. As seen in Fig. 3A, increased expression To assess the Ag presentation ability of transgenic DCs, we of the early activation marker, CD69, did not occur in T cells b bϪ Ϫ asked whether BMDCs could present OVA323Ð339 peptide to I-A - immunized in I-A␤ / hosts, thus the transferred OTII cells restricted OVA-specific OTII TCR-transgenic CD4ϩ T cells (25). were not contaminated by functional class II-positive cells. In con- As expected, OTII division was MHCII dependent, since trast, equivalent numbers of CD69-positive OTII CD4ϩ T cells b b b I-A␤ Ϫ/Ϫ BMDCs failed to induce proliferation at all Ag doses were observed in CD11c/A␤ and I-A␤ ϩ/Ϫ mice on days 1, 2, 3, (Fig. 2, B and C). Over a broad range of either peptide concentra- 4, and 7 postimmunization (Fig. 3A and data not shown). By day b tion or DC number, the frequency of OTII cells responding to 4 postimmunization, OTII cells primed in CD11c/A␤ and b b b Ag-loaded CD11c/A␤ DCs and wild-type BMDCs was equivalent I-A␤ ϩ/Ϫ mice, but not I-A␤ Ϫ/Ϫ hosts, exhibited a similar (Fig. 3B). Similar results were obtained when DCs were loaded CD62Llow CD45RBlowCD44high effector phenotype. These results with OVA protein, indicating intact Ag processing (data not shown). indicated that in vivo differentiation and activation occur normally b Thus, transgenic expression of I-A␤ does not alter DC maturation when MHCII is restricted to DCs. b and restores the Ag presentation function of I-A␤ Ϫ/Ϫ DCs. Proliferation kinetics were assessed by CFSE dye dilution and by determining the number of OTII cells present in various organs. Ag presentation by CD8␣ and CD11b DCs drives differentiation ϩ ϩ Naive OTII CD4 T cells fail to undergo homeostatic proliferation of Ag-specific CD4 T cells in vivo (41) and T cell division required immunization with cognate pep- b Given that bone marrow-derived CD11c/A␤ DCs stimulated nor- tide (Fig. 3B, top panel). Importantly, as with CD69 expression,

b Downloaded from mal proliferative responses, we wished to determine whether DCs OTII cells did not proliferate in I-A␤ Ϫ/Ϫ mice. In contrast, DC are sufficient for naive CD4ϩ T cell primary responses in vivo. Ag presentation was sufficient for primary expansion to peptide Since MHCII-restricted CD4ϩ T cells do not develop in CD11c/ immunizations, as the proliferative profile of responder OTII cells b b b A␤ mice, we examined the responses of congenic CFSE-labeled in CD11c/A␤ and I-A␤ ϩ/Ϫ mice was similar throughout the first OTII CD4ϩ T cells transferred into naive hosts. The proliferation 7 days after immunization (Fig. 3B). Fewer OTII cells committed and differentiation of OTII CD4ϩ T cells following i.v. immuni- to division in both genotypes following immunization with a lower

␮ http://www.jimmunol.org/ zation with OVA323Ð339 peptide with LPS or the adjuvant alone dose of Ag (25 g), but the division proﬁles were again equivalent b b were monitored. in CD11c/A␤ and I-A␤ ϩ/Ϫ mice (data not shown). Remarkably, by guest on September 25, 2021

FIGURE 3. CD8␣ϩ and CD11bϩ DCs are sufﬁcient for primary CD4 T cell responses after peptide immunization. A, Phenotypic markers of OTII cells (VB5ϩCD4ϩCD45.1ϩ) 4 days af- ϭ ter OVA323Ð339 plus LPS immunization (n 4). B, CFSE plots of transferred OTII cells recovered at days 2, 4, and 7 from mice immunized ϭ i.v. with LPS or OVA323Ð339 plus LPS (n 3). Representative plots from LPS immunizations are from day 4, but resemble other days tested. C, Kinetics of OTII cell recovery after

OVA323Ð339 plus LPS immunization. Fold in- crease represents the ratio of OTII cell numbers recovered from immunized mice compared with mice that received LPS alone. SE represents cumulative experiments with two to nine mice per data point. D, Intracellular cytokine staining from naive OTII cells and b OTII cells primed 4 or 7 days in CD11c/A␤ bϩ Ϫ or I-A␤ / mice with OVA323Ð339 plus LPS (n ϭ 3). E, OTII cell recovery from lungs of immunized mice at day 7 postimmunization with OVA323Ð339 plus LPS or LPS alone. 5082 DC Ag PRESENTATION CONTROLS CD4ϩ Th1 CELL RESPONSES

1 and 2 were remarkably similar (Fig. 3B and data not shown). It is possible that, in the absence of endogenous CD4ϩ T cells in b CD11c/A␤ mice, activated OTII T cells can divide further. In- deed, preliminary experiments suggest that OTII division in b b I-A␤ ϩ/Ϫ and CD11c/A␤ mice with full CD4 T cell compartments is identical (M.P.L. and T.M.L., unpublished data), and that the slightly enhanced proliferation could be a product of T cell competition for DC niches. To examine Th1 differentiation, primed OTII CD4ϩ T cells were restimulated in vitro and intracellular IL-2 and IFN-␥ were analyzed. Because OTII cells could not be recovered from b ϩ I-A␤ Ϫ/Ϫ mice in sufﬁcient numbers, naive OTII CD4 T cells served as unprimed controls. At days 4 and 7 after immunization, equivalent percentages of OTII CD4ϩ T cells primed in CD11c/ b b A␤ and I-A␤ ϩ/Ϫ mice produced IL-2 (Fig. 3D). IFN-␥ production could not be detected until day 7 after stimulation. A slightly increased percentage of Ag-speciﬁc OTII cells stimulated in b CD11c/A␤ mice produced IFN-␥; this may correlate with the in-

creased CFSE dilution observed in Fig. 3B since Th1 polarization Downloaded from is cell-cycle dependent (42). Following immunization with Ag and LPS, no IL-4 could be detected at any time point in any of the hosts (data not shown). In conclusion, Ag presentation by CD11bϩ and CD8␣ϩ DCs was sufﬁcient for Th1 differentiation of naive OTII cells, which occurred with normal kinetics. ϩ

Effector CD4 T cells primed to produce IFN-␥ migrate out of http://www.jimmunol.org/ the lymphoid organs and into peripheral tissues (43). Therefore, we examined the accumulation of primed OTII CD4ϩ T cells in the lungs and thymi of immunized mice. Similar numbers of OTII cells migrated into the lungs (Fig. 3E) and thymi (data not shown) b b of CD11c/A␤ and I-A␤ ϩ/Ϫ mice after immunization with

OVA323Ð339 and LPS. Thus, expression of MHCII limited to DCs is sufficient for migration of activated cells into peripheral tissues. I-E␣ peptide-restricted CD4ϩ T cells purified from TEa mice (24) also responded equivalently to i.v. peptide immunization in by guest on September 25, 2021 b b CD11c/A␤ and I-A␤ ϩ/Ϫ mice (data not shown). This result indicated that our findings could be extrapolated to CD4ϩ T cells with different affinities and specificities. In vivo presentation of protein Ags Peptide immunizations bypass internalization and Ag processing within APCs and might favor exogenous loading of Ag on DCs FIGURE 4. CD8␣ϩ and CD11bϩ DCs are sufficient for primary CD4 T (44). Therefore, we verified that OTII T cell differentiation in cell responses after OVA protein immunization. A, CFSE plots of trans- b ferred OTII cells recovered at days 4 and 7 from mice immunized i.v. with CD11c/A␤ mice also occurred following immunization with LPS or OVA plus LPS (n ϭ 3). Representative plots from LPS immuni- OVA protein rather than OVA323Ð339 peptide. As with peptide zations are from day 4, but resemble other days tested. B, OTII cell re- immunizations, we followed OTII cell activation, proliferation of covery after OVA plus LPS immunization (n ϭ 4) expressed as ratio of OTII cells in the spleen and LNs, and migration. Additionally, OTII cell numbers from immunized mice compared with mice that re- anti-OVA IgG Abs in the serum were assayed as a measure of T ceived LPS alone. C, Anti-OVA IgG titers in serum 13 days after immu- cell help to B cells. nization with OVA plus LPS or LPS alone. As with peptide immunizations, division of OTII CD4ϩ T cells in response to protein immunizations required I-Ab expression; no b division or activation was apparent in I-A␤ Ϫ/Ϫ mice (Fig. 4A). the total number of OTII cells recovered from the spleen and LNs The division profiles of OTII CD4ϩ T cells from spleen and LNs b b b b of CD11c/A␤ and I-A␤ ϩ/Ϫ mice was statistically equivalent for stimulated in CD11c/A␤ and wild-type I-A␤ ϩ/Ϫ mice were b 13 days following immunization (Fig. 3C). These results indicated identical on days 4 and 7 (Fig. 4A). In CD11c/A␤ mice, OTII that Ag presentation by DCs is capable of generating all of the expansion and contraction occurred similarly to that in host ani- effector cells during the primary immune response; Ag presenta- mals with normal APC compartments (Fig. 4B). Additionally, ac- tion by other APCs was necessary for neither the expansion of tivated OTII CD4ϩ T cells assumed effector/memory phenotypes Ag-specific effectors nor the contraction phase that follows. and migrated into the lungs (data not shown). The results indicated Interestingly, in some experiments activation of OTII T cells in that Ag processing and presentation of protein Ags by DCs medi- b CD11c/A␤ mice led to a slight accumulation of more highly diated priming, proliferation, differentiation, and migration of OTII vided cells at days 4 and 7 after immunization even though the cells in vivo in the absence of MHCII in other APCs. recovery of OTII cells was comparable (Fig. 3, B and C). This was Because isotype switching requires cognate interactions be- not due to an earlier onset of proliferation, as the CFSE profiles of tween activated CD4 T cells and Ag-specific B cells (14, 45), the b b OTII cells purified from CD11c/A␤ and I-A␤ ϩ/Ϫ mice at days level of anti-OVA IgG in the serum 13 days after immunization The Journal of Immunology 5083 was measured. As expected, no OVA-specific IgG was detected in After expansion in LNs, activated CD4ϩ T cells migrate into the absence of cognate Ag or MHCII, whereas wild-type nonlymphoid organs and accumulate in inflamed sites (43). In vitro b I-A␤ ϩ/Ϫ mice immunized with OVA protein and LPS had ele- data have shown that integrin expression in activated cells is in- vated titers of specific Abs. OTII T cells primed in TCR␣Ϫ/Ϫ mice duced early during the priming of CD4ϩ T cells (51). Our results (23) drove anti-OVA IgG production, indicating OTII cells alone indicate interactions with DCs are sufficient for the modulation of can drive isotype switching in the context of intact MHCII expres- chemokine and adhesion receptors that allow effector cells to exit ϩ b sion (Fig. 4C). The primed OTII CD4 T cells in CD11c/A␤ the LNs and reach peripheral sites in vivo. Studies from Jenkins mice, however, were unable to deliver help to MHCII-negative B and his colleagues (20) have elegantly demonstrated that effector cells. This result confirmed the requirement of cognate interactions CD4ϩ T cells can migrate into inflamed tissue in the absence of between CD4ϩ T cells and B cells for specific IgG production and cognate Ag in a CD62P-dependent manner. Our results support verified the absence of functional MHCII expression in the B cells their conclusions and suggest that if there is a requirement for b of CD11c/A␤ mice. Thus, these results indicated that DC Ag pro- MHCII, DCs are sufficient to mediate these interactions; neither cessing and presentation during the primary response are sufficient endothelial nor parenchymal MHCII expression is required for this for OTII T cell differentiation into effector cells, but fail to direct migration. isotype switching. During the primary response, Ag-specific T cell numbers in lymphoid organs are carefully regulated by the cytokine environ- ment, migration into peripheral tissues, cell death, and competition Discussion between T cell clones. After i.v. peptide and protein immuniza- ϩ ϩ

b b Downloaded from We have restored I-A␤ expression in the CD11b and CD8␣ tions, we observed no differences in T cell recovery in CD11c/A␤ b b b DCs of I-A␤ Ϫ/Ϫ mice, permitting us to compare the immune vs I-A␤ ϩ/Ϫ mice, even though CD11c/A␤ mice lack endoge- b ϩ b response of I-A -restricted Th1 CD4 T cells in hosts with wild- nous MHCII-restricted CD4 T cells and I-A␤ ϩ/Ϫ mice have type- or DC-restricted MHCII distribution. I-Ab expression is ap- full T cell compartments. Our results suggest that during the pri- propriately regulated during DC maturation, and these transgenic mary response, DC cognate interactions with CD4 T cells must DCs can mediate all aspects of Th1 immunity. regulate clonal competition for Ag and expansion. Similarly, our

It is widely assumed that DCs initiate primary immune re- results indicate that the contraction phase of the response does not http://www.jimmunol.org/ sponses. However, in vivo experiments addressing DC function require cognate interactions with B cells, macrophages, or paren- have often relied on Ag-pulsed mature DCs to initiate T cell ac- chymal cells. If TCR-mediated signaling is required for apoptosis tivation, bypassing DC migration and maturation in vivo. Addi- of activated precursors in vivo, DC Ag presentation can mediate tionally, such studies have not been able to contend with the issue these events. Others have shown that DC Ag presentation is ca- of Ag transfer to other APCs. Moreover, adoptive transfer studies pable of mediating naive CD4ϩ T cell homeostasis (4) and our have been unable to address long-term roles for DCs, as i.v. trans- results extend those findings to the generation, maintenance, and fers of mature DCs are short-lived and have limited ability to repop- contraction of effector CD4ϩ T cell numbers. ulate the host (46). The data reported here demonstrate that MHCII- So what is the role for MHCII Ag presentation by non-DCs? dependent Ag presentation by DCs is sufficient to mediate naive CD4 Clearly, MHCII expression by B cells is required for isotype- by guest on September 25, 2021 T cell priming, Th1 differentiation, and control of effector cell pool switched Ab responses. In vitro experiments suggested that MH- size in vivo after peptide and protein immunizations. CII-negative B cells could receive help from activated CD4ϩ T b Because not all DC subsets express MHCII in CD11c/A␤ mice, cells (52). However, Williams et al. (14) found that MHCII on B this system has allowed us to test the cognate requirements for cells was required for IgG production of T-dependent Ags in vivo, individual DC subsets. Thus, the functions that are intact in even in the context of productive T cell priming. Similarly, using b CD11c/A␤ mice do not require Ag presentation by LCs or pDCs, a chimeric approach, Fillatreau and Gray (45) demonstrated that, as these subsets lack MHCII expression. Roles for LCs and pDCs although CD4ϩ T cell migration into B cell zones is independent have been proposed in Th1 immunity (5, 9, 18, 47); however, Th1 of MHCII on B cells, isotype switching in this setting is defective. b effector differentiation and function occurs normally in CD11c/A␤ Our results agree with these in vivo observations, as DC MHCII mice under the current immunization protocol. These results sug- expression cannot overcome the requirement for B cell-T cell cog- gest that putative roles for LCs and pDCs in Th1 primary re- nate interactions. However, this is not to say that B cell Ag pre- sponses can be independent of MHCII expression. It is possible sentation cannot affect the T cell response, since a role for B cells that OVA immunizations with LPS, which signals through Toll- in Th2 differentiation, CD4ϩ T cell memory responses, and pe- like receptor 4 on the surface of CD11bϩ DCs and CD8␣ϩ DCs ripheral tolerance has been previously reported (12, 13, 49, 50). (21), bypass pDCs which express low levels of Toll-like receptor Finally, is there a role for macrophage and parenchymal MHCII 4 (8). On the other hand, i.v. delivery of Ag might bypass a role for expression? The roles of MHCII presentation could be more im- LCs in peptide and protein immunizations. Future experiments will portant during the effector phase of the response rather than Th1 determine whether there is a requirement for pDCs and LCs using polarization. We are currently addressing the sufficiency of DC other methods of immunization. MHCII Ag presentation in the effector phases of the T cell re- The normal primary immune response is characterized by the sponse. However, it has been suggested that Ag presentation by selection and expansion of CD4ϩ T cell precursors and their dif- human endothelial cells facilitates the migration of Ag-specific ferentiation into effector cells. Immunohistochemistry studies by cells into the tissues (19). Our findings and others indicate that Attinger et al. (48) suggested that most T cell proliferation occurs neither Ag (20) nor MHCII expression by parenchymal cells is at the T cell zones. In agreement with their results, our findings required for the migration of Ag-specific murine CD4ϩ T cells into indicate that naive T cells can rely exclusively on DCs, the major peripheral sites. However, parenchymal MHCII presentation might APC in the T cell zones, for Ag-driven expansion. It remains to be play a role in the long-term maintenance of Ag-specific lympho- addressed whether MHCII expression on B cells, macrophages, cytes at sites of infection, which was not addressed by the current b and parenchymal tissues plays a more important role in Th2 dif- studies. Further study of the CD11c/A␤ mouse model will allow ferentiation, where a continuous low level of Ag is considered us to delineate the distinct roles of different APCs in regulating Ag more important than in Th1 differentiation (12, 15, 49, 50). presentation to CD4ϩ T cells during immunity and autoimmunity. 5084 DC Ag PRESENTATION CONTROLS CD4ϩ Th1 CELL RESPONSES

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CORRECTIONS

Koji Uraushihara, Takanori Kanai, Kwibeom Ko, Teruji Totsuka, Shin Makita, Ryoichi Iiyama, Tetsuya Nakamura, and Mamoru Watanabe. Regulation of Murine Inﬂammatory Bowel Disease by CD25ϩ and CD25ϪCD4ϩ Glucocorticoid- Induced TNF Receptor Family-Related Gene Regulatory T Cells. The Journal of Immunology 2003;171:708Ð716.

In the institution afﬁliation footnote, Tokyo Medical and Dental University should have been ﬁrst.

Alon Monsonego, Jaime Imitola, Victor Zota, Takatoku Oida, and Howard L. Weiner. Microglia-Mediated Nitric Oxide Cytotoxicity of T Cells Following Amyloid ␤ Peptide Presentation to Th1 Cells. The Journal of Immunology 2003;171: 2216Ð2224.

In Materials and Methods, under the heading, Preparation of cultures of mouse brain microglis, the concentrations for streptomycin and mercaptoethanol were published incorrectly. The correct sentence is shown below.

Glial cultures were prepared as follows: cells were dissociated from the cerebral cortex of 1-day-old C57BL/6 mice, 2 carefully removing meninges tissue, and were cultured in poly-D-lysine-coated tissue culture ﬂasks (two brains per 85-cm ﬂask) in medium supplemented with DMEM, 4 mM L-glutamine, 50 U/ml penicillin, 50 ␮g/ml streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 10 mM nonessential amino acids, 57.2 ␮M 2-ME (Sigma-Aldrich, St. Louis, MO), and 10% FCS.

Aleksandar K. Stanic, R. Shashidharamurthy, Jelena S. Bezbradica, Naoto Matsuki, Yoshitaka Yoshimura, Sachiko Miyake, Eun Young Choi, Todd D. Schell, Luc Van Kaer, Satvir S. Tevethia, Derry C. Roopoenian, Takashi Yamamura, and Sebastian Joyce. Another View of T Cell Antigen Recognition: Cooperative Engagement of Glycolipid Antigens by Va14Ja18 Natural TCR. The Journal of Immunology 2003;171:4539Ð4551.

The last abbreviation in the article title was published incorrectly. The correct title is shown below.

Another View of T Cell Antigen Recognition: Cooperative Engagement of Glycolipid Antigens by Va14Ja18 Natural T (iNKT) Cell Receptor.

Maria P. Lemos, Lian Fan, David Lo, and Terri M. Laufer, CD8ϩ and CD11bϩ Dendritic Cell-Restricted MHC Class II Controls Th1 CD4ϩ T Cell Immunity. The Journal of Immunology 2003;171:5077Ð5084.

In Materials and Methods, the legends for Figures 1B and 1C were inverted. The legend for Figure 1C referenced the graph in Figure 1B, and vice versa.