HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) A correction has been published Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lemos, M. P. Articles by Laufer, T. M. Search for Related Content PubMed PubMed Citation Articles by Lemos, M. P. Articles by Laufer, T. M. Pubmed/NCBI databases Substance via MeSH

CD8⁺ and CD11b⁺ Dendritic Cell-Restricted MHC Class II Controls Th1 CD4⁺ T Cell Immunity ¹

Maria P. Lemos^*, Lian Fan^2,, David Lo^3, and Terri M. Laufer^4,*

^* Department of Medicine, University of Pennsylvania, Philadelphia PA 19104; The Scripps Research Institute, La Jolla, CA 92037

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 in a mouse model in which I-A^b expression was targeted specifically to the DCs of I-Ab-/- mice. MHCII expression is reconstituted in CD11b⁺ and CD8⁺ DCs, but other DC subtypes, macrophages, B cells, and parenchymal cells lack of expression of the I-A^b 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-. Additionally, proliferation, migration out of lymphoid organs, and the number of effector CD4⁺ T cells are appropriately regulated. 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.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Major histocompatibility complex class II (MHCII)⁵ molecules are required for the development of CD4⁺ T cells in the thymus and function to present peptide Ags to CD4⁺ T cells in the periphery (1, 2). Constitutive expression of MHCII molecules at peripheral sites is limited to professional APCs such as macrophages, dendritic cells (DCs), and B cells. MHCII expression can also be induced on endothelial and parenchymal tissues by inflammatory stimuli such as IFN- (3). Although peripheral MHCII expression on DCs mediates the survival of CD4⁺ T cells (4), the specific role of individual MHCII-positive APCs during CD4⁺ T immune responses has not been fully elucidated.

Priming naive CD4⁺ T cells is largely the function of DCs, which present Ag in the context of high levels of MHC, costimulatory molecules, activating cytokines, and T cell-attracting chemokines (5, 6). DCs can be distinguished from other class II-positive APCs by the expression of the integrin pair CD11c/CD18 (6). CD11c⁺ DCs can be further divided into multiple subsets based on surface markers, anatomic localization, and cytokine production (5). Chief among these subsets are: CD11c^neg-low epidermal Langerhans cells (LCs), CD11b⁺ DCs, CD8⁺ DCs, and CD11c^lowB220⁺Ly6G/C⁺ plasmacytoid DC (pDCs). These DC subsets have all been shown to regulate Th1 priming, but whether they play redundant or specific roles in T cell immune responses is unknown (7, 8, 9). Thus, it is unclear whether cognate interactions with all DC types are necessary for normal Th1 immune responses, or whether individual cell types direct polarization of Th1 cells in a noncognate manner.

Mature DCs have a very short life span (10); therefore, T cell interactions with other APCs may contribute to complete primary immune responses (11). Thus, B cells may contribute to the full expansion of Ag-specific CD4 T cells to drive Th2 differentiation, but whether this requires Ag presentation is controversial (12, 13, 14). However, the effects of B cell Ag presentation on Th1 CD4⁺ differentiation are unknown.

The differentiation and function of Th1 effector cells relies on TCR-MHCII interactions, proliferation, and the cytokine milieu. The strength of the MHCII-TCR signal plays an important role in Th1 polarization (8, 15); therefore, the lack of MHCII from certain APCs could affect the Th1/Th2 balance. MHCII-peptide-TCR interactions facilitate production of IL-12, which drives Th1 polarization, by macrophages and DCs (5, 16, 17, 18); thus, both APCs might be important in the differentiation and function of Th1 effectors. TCR-MHCII interactions also may contribute to the recruitment and accumulation of CD4⁺ T cells into areas of inflammation (19), although recent evidence has suggested this event does not require Ag (20). Therefore, local MHCII⁺ macrophages and parenchymal cells might be required for accumulation of effector cells at infected sites.

Maturation signals to DCs and Ag dose have been reported to play a crucial role in Th differentiation (5, 8, 15, 21). Studies addressing the role of DCs in CD4⁺ T cell differentiation have used DCs that have been matured and loaded with Ag in vitro and then transferred i.v. or s.c. into hosts. We chose to evaluate the sufficiency of MHCII Ag presentation by DCs, allowing Ag uptake, MHCII peptide loading, DC maturation, and migration to occur in vivo. To do so, we used the CD11c promoter to target MHCII expression exclusively to the DCs of class II-deficient mice. We demonstrate that CD8⁺ and CD11b⁺ DCs are sufficient for the expansion, differentiation, migration, and contraction of Ag-specific Th1 CD4⁺ T cells during the primary response. These findings indicate that Ag presentation by DCs not only controls priming events, but regulates the fate of Th1 effector CD4 T cells throughout the response.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Transgenic mice

The CD11c promoter (4) was obtained from Dr. I. Mellman (Yale University, New Haven, CT). The A^b cDNA (22) (gift from Dr. R. Germain, National Institutes of Health, Bethesda, MD) was inserted into the EcoRI cloning site in the CD11c cassette (4). The linearized CD11c/A^b construct was injected into (BALB/c x C57BL/6)F₁ eggs. Three different founder lines were generated and backcrossed to C57BL/6 mice for 6–10 generations.

C57BL/6, C57BL/6J-Tcra^tm1Mom (TCR^-/-) (23), and B6.SJL-Ptpcrc^a Pep3^b/BoyJ (CD45.1) congenics were obtained from The Jackson Laboratory (Bar Harbor, ME). I-A^b-/- mice (2) were bred in our colony (22 generations backcrossed to C57BL/6 background). TEa mice (24), a gift from Dr. M. Jenkins (University of Minnesota, Minneapolis, MN), and OTII mice (25), a gift from Dr. C. Surh (The Scripps Institute, La Jolla, CA), were bred to CD45.1 congenics. Mice 6–10 wk of age were used in all experiments.

Immunohistochemistry

Cryosections of spleen and thymus (5 µm) were fixed in cold acetone, washed in PBS and 0.3% H₂O₂, and blocked with goat serum and avidin-biotin block (Vector Laboratories, Burlingame, CA). Samples were stained with primary Ab against I-A^b/d (M5/114) and CD3 (2C11; American Type Culture Collection (ATCC), Manassas, VA). Adjacent sections were 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-hamster IgG Fabs (Jackson ImmunoResearch, West Grove, PA) followed by the ABC Elite kit or Vectastain ABC Alkaline Phosphatase kit (Vector Laboratories). HRP and alkaline phosphatase were developed using 3-amino-9-ethylcarbazole (Sigma-Aldrich, St. Louis, MO) or Vector Blue alkaline phosphatase kit (Vector Laboratories), respectively.

DC isolation

Organs were dissected, placed in incomplete Iscove’s medium on ice, injected with 1 ml of 5 mg/ml collagenase type IV (Worthington Biochemicals, Lakewood, NJ) and 0.3% DNase I (Sigma-Aldrich), and incubated at room temperature for 30–45 min. Single-cell suspensions were washed and resuspended in PBS with 2% BSA, 2 mM EDTA, polyclonal rat IgG (Sigma-Aldrich), and CD11c MACS beads (Miltenyi Biotec, Auburn, CA). After staining, cells were washed and purified on paramagnetic AUTOMACS (Miltenyi Biotec) columns according to the manufacturer’s instructions. CD11c⁺ cell purity ranged from 70 to 98% depending on the organ.

pDCs were analyzed by flow cytometry after positive selection of CD45R/B220⁺ splenocytes on paramagnetic AUTOMACS columns (Miltenyi Biotec) using anti-CD45R-biotin (BD PharMingen, San Diego, CA) and anti-biotin microbeads (Miltenyi Biotec). LCs were purified from ear skin explants as described before (26, 27). In vitro culture of epidermal and dermal layers was conducted in complete RPMI 1640 with 100 ng/ml recombinant mouse GM-CSF (PeproTech, Rocky Hill, NJ) for 24 h. Migrating LCs collected in the media were analyzed by flow cytometry using anti-CD45 to identify the hemopoietic LCs.

Flow cytometry

Single-cell suspensions were blocked with Abs against FcRII/III (24G2; ATCC). The Abs used for staining were I-A^b-FITC (AF6), rIgG-FITC, CD11c-PE, human IgG2a-PE, CD8-PerCP, rIgG-PerCP, CD11b-allophycocyanin, rIgG-allophycocyanin, CD45-Bio, CD45R-bio, CD86-FITC, CD80-FITC, CD40-FITC, CD45-FITC, CD4-FITC, human IgG-FITC, mouse IgG-FITC, V2-FITC, I-A^b-PE, B220-PE, CD44-FITC, CD62L-FITC, CD45RB-FITC, CD69-FITC, CD69-PE, CD8-FITC, CD4-PerCy, V5-PE, CD45.1-biotin, CD19-allophycocyanin, streptavidin-allophycocyanin, and streptavidin-FITC (BD PharMingen). Samples were analyzed on a FACSCalibur (BD Biosciences, San Diego, CA) using CellQuest software. All dot plots shown have a log axis of 10⁰–10⁴. Staining of macrophages, DCs, and activated B cells was conducted in PBS with 2% BSA, 2 mM EDTA, 1 µg/ml mouse IgG (Sigma-Aldrich), and 1 µg/ml rat IgG (Sigma-Aldrich). Macrophage analysis excluded CD11c^highCD86^high DCs. Intracellular MHCII was performed on 1% paraformaldehyde-fixed samples that were permeabilized with PBS/2% BSA/0.02% saponin.

For intracellular cytokine staining, CD45.1⁺ cells were purified using CD45.1 biotin (BD PharMingen) and anti-biotin microbeads (Miltenyi Biotec) on paramagnetic columns. Cells were treated with 50 ng/ml PMA and 500 ng/ml Ionomycin in the presence of 2 nM monensin for 5 h at 37°C. OTII cells (V2 V5) were surface stained for V5, CD4, and CD45.1, followed by fixation and permeabilization as described above. Intracellular cytokine staining was performed using anti-IL-2-allophycocyanin, IL-4-allophycocyanin, and IFN--allophycocyanin (BD PharMingen).

B cell and macrophage stimulation

To obtain activated B cells, splenocytes were cultured for 48 h in DMEM with 10% FBS, 50 µg/ml LPS from Salmonella typhimurium (Sigma-Aldrich), and 10 ng/ml recombinant mouse IL- 4 (PeproTech). Peritoneal macrophages were harvested in 10 ml of 10% FBS in RPMI 1640 and cultured with 100 U/ml IFN- (PeproTech) for 72 h.

Bone marrow-derived DCs (BMDC)

BMDCs were generated as previously described (28) using recombinant mouse GM-CSF (PeproTech) or supernatants of B78H1/GM-CSF cell line cultures, a kind gift from Dr. H. Levitzky (Johns Hopkins University, Baltimore, MD) in the BMDC cultures for 8 days. For BMDC maturation, 1 µg/ml LPS was added 18 h before analysis of CD11c⁺CD11b⁺ cells. Student’s paired t test was used to determine statistical significance in the maturation markers and MHCII levels.

For peptide-pulsed BMDCs, on day 6, OVA peptide 323–339 (University of Pennsylvania Cancer Center Peptide Synthesis Core) was added to the cultures in concentrations ranging from 0.01 to 3 µg/ml; OVA protein (Sigma-Aldrich) was added in concentrations ranging from 100 to 1 µg/ml. Ag-pulsed BMDCs were washed three times and cultured with 1 x 10⁵ CFSE-labeled OTII CD4⁺ T cells for 4 days.

OTII CD4 T cell purification, priming, and analysis

OTII CD4⁺ T cells were purified by positive selection of CD4⁺ cells on AUTOMACS columns (Miltenyi Biotec) and were 94–98% V2⁺. CFSE (Molecular Probes, Eugene, OR) labeling of OTII cells and quantitation of CFSE dilutions were conducted according to previously published protocols (29, 30). Responder frequency is the proportion of the initial pool that divides after stimulation (30).

For in vivo experiments, three to five million OTII CD4⁺ T cells were transferred i.v. into each recipient. Twenty-four hours later, mice were immunized i.v. (31) with 100 µg OVA_323–339 and 75 µg LPS, 300 µg OVA protein (Sigma-Aldrich), and 75 µg LPS or 75 µg LPS (Sigma-Aldrich).

To analyze OTII activation, lungs, spleens, and lymph nodes (LNs) were analyzed by flow cytometry. Lungs from immunized mice were cultured in 10 ml of 0.5 mg/ml collagenase type IV (Worthington Biochemicals) and 0.1% DNase I (Sigma-Aldrich) and incubated at 37°C for 30–45 min. After digestion, lungs were dissociated into single-cell suspensions and separated on Percoll gradients (32). OTII CD4⁺ T cell recovery is based on the number of CD45.1⁺CD4⁺V5⁺ cells found in the preparations of LNs and spleen. Statistical tests for OTII recovery differences used Student’s paired t and Mann-Whitney U tests.

ELISA

Anti-OVA IgG was measured by ELISAs as previously described (33) using diluted serum samples. IgG titers were determined using alkaline phosphatase-conjugated goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL) and developed as previously described (53).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD8⁺ and CD11b⁺ DCs are I-A^b positive in CD11c/A^b mice

To probe the sufficiency of MHCII-dependent Ag presentation mediated by DCs, we have examined the phenotype of transgenic mice in which I-A^b expression is restricted to DCs. Brocker (4) previously reported that the murine CD11c promoter reconstitutes I-E expression in 70% of thymic and splenic DCs in transgenic C57BL/6 mice. We used this promoter to selectively re-express the I-A^b chain in the I-A^b-/- (MHCII-deficient) background. Two founder lines led to MHCII expression on 12 and 40% of DCs; a third founder line had 90% of wild-type MHCII⁺ DCs and was used for all experiments.

Transgene-positive mice were backcrossed to I-A^b-/- mice (CD11c/A^b mice) and analyzed for restoration of class II expression. Surface expression of I-A^b in CD11c/A^b mice should occur in cells where transgenic I-A^b expression overlaps with endogenous I-A^b expression. To quantitate the expression of I-A^b induced by the transgene, we performed FACS analysis on DCs obtained from multiple organs. Equivalent numbers of DCs were obtained from I-A^b-/-, CD11c/A^b, and I-A^b+/- mice, supporting previous observations that MHC class II expression is not required for normal DC development and localization (15, 34). In lymphoid organs, >90% of CD11c/A^b DCs had surface MHCII expression greater than I-A^b-/--negative controls (Fig. 1A). Thus, the CD11c/A^b transgene rescued MHCII expression on most DCs within lymphoid organs.

View larger version (63K): [in this window] [in a new window]   FIGURE 1. Characterization of MHCII expression in CD11c/Ab mice. M1 refers to staining above I-Ab-/- controls. A, Total CD11c+ DCs from spleen and LNs (n = 8). B, CD8+ DCs (n = 3). C, CD11b+ DCs (n = 5). D, CD19-B220+CD11clow pDCs (n = 3). E, LCs (CD45+ cells migrating out of skin explants) (n = 4). F, Immunohistochemistry of thymus sections stained with Abs against I-Ab (M5114). Representative photographs (n = 3). C, Cortex; M, medulla. G, Immunohistochemistry of the spleen stained with Abs against I-Ab (red) and CD3 (blue). Representative photographs (n = 3). T, T cell zones; B, B cell zones. H, intracellular MHCII on B220highCD19+ B cells (n = 3). I, Surface MHCII on LPS-treated B cells (n = 5). J, Surface MHCII on IFN--treated peritoneal macrophages (n = 4).

We expected that DC subsets with low levels of CD11c, mainly pDCs and LCs, would remain I-A^b negative and this was the case. CD19^-B220⁺CD11c^low pDCs lacked significant MHC class II expression in CD11c/A^b mice, whereas I-A^b+/- pDCs are I-A^b/low (Fig. 1D). In agreement with a recent report that this promoter cassette did not drive expression in LCs (35), skin-resident Langerhans’ cells were also I-A^b negative and remained class II-negative following epidermal irritation and migration into the draining LN (Fig. 1E and data not shown). Therefore, we suspect that the 10% of lymphoid organ DCs that remain class II-negative in CD11c/A^b mice (Fig. 1A) include pDCs, LCs, and other CD11c^low subsets.

Immunohistochemistry was used to examine the localization of the transgenic class II-positive DCs in lymphoid organs. In the thymus, anti-I-A^b staining was detected on scattered stellate cells in the medulla (Fig. 1F). These cells were also CD11c positive (data not shown). Because CD11c-driven transgenes do not drive expression in thymic epithelium, the pattern of MHCII expression in CD11c/A^b mice fails to drive positive selection of MHCII-restricted cells (data not shown), and CD11c/A^b mice have no more CD4⁺ T cells than do I-A^b-/- mice (36).

Others have previously reported that splenic morphology is independent of MHCII expression and the presence of T cells (15, 34, 37). In agreement with these reports, CD11c/A^b mice had well-defined B cell and T cell zones (Fig. 1F). I-A^b expression in CD11c/A^b spleens localized to the T cell zones and scattered areas of the red pulp and paralleled anti-CD11c staining of adjacent sections (data not shown). No I-A^b staining of B cell areas was detected in CD11c/A^b spleens. Thus, CD11c/A^b mice maintain normal splenic architecture and MHCII⁺ DCs are appropriately localized.

Finally, we analyzed resident tissue DC isolated from liver and lungs. DCs purified from CD11c/A^b lungs expressed equivalent levels of I-A^b when compared with I-A^b+/- lung DCs; in contrast, only a quarter of the DCs present in the liver of CD11c/A^b mice were MHCII positive (data not shown). The MHCII expression pattern correlated with the different DC subsets present in these organs: lungs have mainly CD11b^-CD8⁺ DCs, whereas the liver contains a higher proportion of pDCs and CD11b⁺CD11c⁺ DCs (38, 39, 40). In conclusion, CD11c-driven I-A^b expression led to complete reconstitution of MHCII in CD8⁺ DCs and slightly decreased I-A^b expression in CD11b⁺ DCs; pDCs and LCs remained MHCII negative.

B cells and macrophages are class II negative in CD11c/A^b mice

To determine whether transgenic I-A^b expression was limited to DCs, B cells and macrophages were analyzed by flow cytometry. Resting B220⁺ splenic B cells and CD11b⁺ peritoneal macrophages lacked surface expression of I-A^b heterodimers. Surface and intracellular staining with anti-A^b-specific Abs (AF6) revealed no more protein in CD11c/A^b B cells than in I-A^b-/- B cells (Fig. 1G and data not shown). To ensure that this phenotype was stable with activation, splenic B cells were cultured with IL-4 and LPS to generate activated B lymphoblasts and then reanalyzed. B cells from all genotypes had equivalent up-regulation of CD69 and CD86, indicating they were activated (data not shown). However, at least 99% of the CD11c/A^b B cells lacked surface I-A^b (Fig. 1H) and intracellular I-A^b expression (data not shown). Finally, LPS-treated B cells could stimulate neither peptide-specific nor allogeneic CD4⁺ T cells responses (data not shown), verifying that these APCs are functionally MHCII negative.

To analyze MHCII expression in activated macrophages, peritoneal macrophages were treated with IFN- for 72 h and the CD11b^highCD11c^low macrophages were analyzed by flow cytometry. Macrophages from all genotypes were activated with increased surface levels of CD80 and CD86 (data not shown). Wild-type I-A^b+/- macrophages increased surface levels of I-A^b; no change was apparent in the level of I-A^b on activated macrophages from CD11c/A^b mice, which remained MHCII negative (Fig. 1I). Therefore, CD11c/A^b mice have MHCII expression restricted to DCs.

CD11c/A^b DCs mature normally and present Ag in vitro

The maturation phenotype (CD40, CD80, CD86) and DC yields of DCs isolated ex vivo from I-A^b+/- mice and CD11c/A^b mice were comparable (data not shown), suggesting that DC maturation was not altered by the presence of the transgene. We verified that DC I-A^b expression was regulated appropriately during maturation in CD11c/A^b mice by following the LPS-driven maturation of BMDCs. Immature BMDCs from all genotypes had comparable levels of CD80, CD86, and CD40 (Fig. 2A, black lines) and these increased equivalently following LPS-induced maturation (Fig. 2A, gray histograms). However, I-A^b expression (mean fluorescence intensity (MFI)) in immature CD11c/A^b BMDCs was two-thirds that of immature I-A^b+/- BMDCs (p = 0.002). Immature BMDCs from CD11c/A^b mice resembled freshly isolated CD11b⁺ DCs and 5–10% of the population also lacked surface MHCII. Importantly, LPS-dependent maturation up-regulated I-A^b in CD11c/A^b BMDCs to levels similar to those of wild-type I-A^b+/- BMDCs (ratio MFI_CD11c/Ab/MFI_I-Ab+/- = 0.9 ± 0.37). Thus, CD11c-driven I-A^b expression is sufficient for wild-type I-A^b surface expression of MHCII heterodimers in mature DCs.

View larger version (33K): [in this window] [in a new window]   FIGURE 2. BMDCs from CD11c/Ab mice have normal maturation and peptide presentation. A, Maturational markers of BMDCs (n = 4). B and C, OTII CD4 T cell proliferation quantitated by CFSE responder frequency (n = 3) in response to different concentrations of OVA323–339 peptide on BMDCs (B) or different numbers of BMDCs pulsed with 0.5 µg/ml OVA323–339 (C).

View larger version (48K): [in this window] [in a new window]   FIGURE 3. CD8+ and CD11b+ DCs are sufficient for primary CD4 T cell responses after peptide immunization. A, Phenotypic markers of OTII cells (VB5+CD4+CD45.1+) 4 days after OVA323–339 plus LPS immunization (n = 4). B, CFSE plots of transferred OTII cells recovered at days 2, 4, and 7 from mice immunized i.v. with LPS or OVA323–339 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 OVA323–339 plus LPS immunization. Fold increase 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 OTII cells primed 4 or 7 days in CD11c/Ab or I-Ab+/- mice with OVA323–339 plus LPS (n = 3). E, OTII cell recovery from lungs of immunized mice at day 7 postimmunization with OVA323–339 plus LPS or LPS alone.

Ag presentation by CD8 and CD11b DCs drives differentiation of Ag-specific CD4⁺ T cells in vivo

Given that bone marrow-derived CD11c/A^b DCs stimulated normal proliferative responses, we wished to determine whether DCs are sufficient for naive CD4⁺ T cell primary responses in vivo. Since MHCII-restricted CD4⁺ T cells do not develop in CD11c/A^b mice, we examined the responses of congenic CFSE-labeled OTII CD4⁺ T cells transferred into naive hosts. The proliferation and differentiation of OTII CD4⁺ T cells following i.v. immunization with OVA_323–339 peptide with LPS or the adjuvant alone were monitored.

We first followed changes in phenotypic markers of activation following immunization. As seen in Fig. 3A, increased expression of the early activation marker, CD69, did not occur in T cells immunized in I-A^b-/- hosts, thus the transferred OTII cells were not contaminated by functional class II-positive cells. In contrast, equivalent numbers of CD69-positive OTII CD4⁺ T cells were observed in CD11c/A^b and I-A^b+/- mice on days 1, 2, 3, 4, and 7 postimmunization (Fig. 3A and data not shown). By day 4 postimmunization, OTII cells primed in CD11c/A^b and I-A^b+/- mice, but not I-A^b-/- hosts, exhibited a similar CD62L^low CD45RB^lowCD44^high effector phenotype. These results indicated that in vivo differentiation and activation occur normally when MHCII is restricted to DCs.

Proliferation kinetics were assessed by CFSE dye dilution and by determining the number of OTII cells present in various organs. Naive OTII CD4⁺ T cells fail to undergo homeostatic proliferation (41) and T cell division required immunization with cognate peptide (Fig. 3B, top panel). Importantly, as with CD69 expression, OTII cells did not proliferate in I-A^b-/- mice. In contrast, DC Ag presentation was sufficient for primary expansion to peptide immunizations, as the proliferative profile of responder OTII cells in CD11c/A^b and I-A^b+/- mice was similar throughout the first 7 days after immunization (Fig. 3B). Fewer OTII cells committed to division in both genotypes following immunization with a lower dose of Ag (25 µg), but the division profiles were again equivalent in CD11c/A^b and I-A^b+/- mice (data not shown). Remarkably, the total number of OTII cells recovered from the spleen and LNs of CD11c/A^b and I-A^b+/- mice was statistically equivalent for 13 days following immunization (Fig. 3C). These results indicated that Ag presentation by DCs is capable of generating all of the effector cells during the primary immune response; Ag presentation by other APCs was necessary for neither the expansion of Ag-specific effectors nor the contraction phase that follows.

Interestingly, in some experiments activation of OTII T cells in CD11c/A^b mice led to a slight accumulation of more highly divided cells at days 4 and 7 after immunization even though the recovery of OTII cells was comparable (Fig. 3, B and C). This was not due to an earlier onset of proliferation, as the CFSE profiles of OTII cells purified from CD11c/A^b and I-A^b+/- mice at days 1 and 2 were remarkably similar (Fig. 3B and data not shown). It is possible that, in the absence of endogenous CD4⁺ T cells in CD11c/A^b mice, activated OTII T cells can divide further. Indeed, preliminary experiments suggest that OTII division in I-A^b+/- and CD11c/A^b 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 CD4⁺ T cells were restimulated in vitro and intracellular IL-2 and IFN- were analyzed. Because OTII cells could not be recovered from I-A^b-/- mice in sufficient numbers, naive OTII CD4⁺ T cells served as unprimed controls. At days 4 and 7 after immunization, equivalent percentages of OTII CD4⁺ T cells primed in CD11c/A^b and I-A^b+/- 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 CD11c/A^b mice produced IFN-; this may correlate with the increased CFSE dilution observed in Fig. 3B since Th1 polarization 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 CD11b⁺ and CD8⁺ DCs was sufficient for Th1 differentiation of naive OTII cells, which occurred with normal kinetics.

Effector CD4⁺ T cells primed to produce IFN- migrate out of the lymphoid organs and into peripheral tissues (43). Therefore, we examined the accumulation of primed OTII CD4⁺ 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) of CD11c/A^b and I-A^b+/- mice after immunization with OVA_323–339 and LPS. Thus, expression of MHCII limited to DCs is sufficient for migration of activated cells into peripheral tissues.

I-E peptide-restricted CD4⁺ T cells purified from TEa mice (24) also responded equivalently to i.v. peptide immunization in CD11c/A^b and I-A^b+/- mice (data not shown). This result indicated that our findings could be extrapolated to CD4⁺ 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 (44). Therefore, we verified that OTII T cell differentiation in CD11c/A^b mice also occurred following immunization with OVA protein rather than OVA_323–339 peptide. As with peptide immunizations, we followed OTII cell activation, proliferation of OTII cells in the spleen and LNs, and migration. Additionally, anti-OVA IgG Abs in the serum were assayed as a measure of T cell help to B cells.

As with peptide immunizations, division of OTII CD4⁺ T cells in response to protein immunizations required I-A^b expression; no division or activation was apparent in I-A^b-/- mice (Fig. 4A). The division profiles of OTII CD4⁺ T cells from spleen and LNs stimulated in CD11c/A^b and wild-type I-A^b+/- mice were identical on days 4 and 7 (Fig. 4A). In CD11c/A^b mice, OTII expansion and contraction occurred similarly to that in host animals with normal APC compartments (Fig. 4B). Additionally, activated OTII CD4⁺ T cells assumed effector/memory phenotypes and migrated into the lungs (data not shown). The results indicated that Ag processing and presentation of protein Ags by DCs mediated priming, proliferation, differentiation, and migration of OTII cells in vivo in the absence of MHCII in other APCs.

View larger version (15K): [in this window] [in a new window]   FIGURE 4. CD8+ and CD11b+ DCs are sufficient for primary CD4 T cell responses after OVA protein immunization. A, CFSE plots of transferred OTII cells recovered at days 4 and 7 from mice immunized i.v. with LPS or OVA plus LPS (n = 3). Representative plots from LPS immunizations are from day 4, but resemble other days tested. B, OTII cell recovery after OVA plus LPS immunization (n = 4) expressed as ratio of OTII cell numbers from immunized mice compared with mice that received LPS alone. C, Anti-OVA IgG titers in serum 13 days after immunization with OVA plus LPS or LPS alone.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have restored I-A^b expression in the CD11b⁺ and CD8⁺ DCs of I-A^b-/- mice, permitting us to compare the immune response of I-A^b-restricted Th1 CD4 T cells in hosts with wild-type- or DC-restricted MHCII distribution. I-A^b expression is appropriately regulated during DC maturation, and these transgenic DCs can mediate all aspects of Th1 immunity.

It is widely assumed that DCs initiate primary immune responses. However, in vivo experiments addressing DC function have often relied on Ag-pulsed mature DCs to initiate T cell activation, bypassing DC migration and maturation in vivo. Additionally, such studies have not been able to contend with the issue of Ag transfer to other APCs. Moreover, adoptive transfer studies have been unable to address long-term roles for DCs, as i.v. transfers of mature DCs are short-lived and have limited ability to repopulate the host (46). The data reported here demonstrate that MHCII-dependent Ag presentation by DCs is sufficient to mediate naive CD4 T cell priming, Th1 differentiation, and control of effector cell pool size in vivo after peptide and protein immunizations.

Because not all DC subsets express MHCII in CD11c/A^b mice, this system has allowed us to test the cognate requirements for individual DC subsets. Thus, the functions that are intact in CD11c/A^b mice do not require Ag presentation by LCs or pDCs, as these subsets lack MHCII expression. Roles for LCs and pDCs have been proposed in Th1 immunity (5, 9, 18, 47); however, Th1 effector differentiation and function occurs normally in CD11c/A^b mice under the current immunization protocol. These results suggest that putative roles for LCs and pDCs in Th1 primary responses can be independent of MHCII expression. It is possible that OVA immunizations with LPS, which signals through Toll-like receptor 4 on the surface of CD11b⁺ DCs and CD8⁺ DCs (21), bypass pDCs which express low levels of Toll-like receptor 4 (8). On the other hand, i.v. delivery of Ag might bypass a role for LCs in peptide and protein immunizations. Future experiments will determine whether there is a requirement for pDCs and LCs using other methods of immunization.

The normal primary immune response is characterized by the selection and expansion of CD4⁺ T cell precursors and their differentiation into effector cells. Immunohistochemistry studies by Attinger et al. (48) suggested that most T cell proliferation occurs at the T cell zones. In agreement with their results, our findings indicate that naive T cells can rely exclusively on DCs, the major APC in the T cell zones, for Ag-driven expansion. It remains to be addressed whether MHCII expression on B cells, macrophages, and parenchymal tissues plays a more important role in Th2 differentiation, where a continuous low level of Ag is considered more important than in Th1 differentiation (12, 15, 49, 50).

After expansion in LNs, activated CD4⁺ T cells migrate into nonlymphoid organs and accumulate in inflamed sites (43). In vitro data have shown that integrin expression in activated cells is induced early during the priming of CD4⁺ T cells (51). Our results indicate interactions with DCs are sufficient for the modulation of chemokine and adhesion receptors that allow effector cells to exit the LNs and reach peripheral sites in vivo. Studies from Jenkins and his colleagues (20) have elegantly demonstrated that effector CD4⁺ T cells can migrate into inflamed tissue in the absence of cognate Ag in a CD62P-dependent manner. Our results support their conclusions and suggest that if there is a requirement for MHCII, DCs are sufficient to mediate these interactions; neither endothelial nor parenchymal MHCII expression is required for this migration.

During the primary response, Ag-specific T cell numbers in lymphoid organs are carefully regulated by the cytokine environment, migration into peripheral tissues, cell death, and competition between T cell clones. After i.v. peptide and protein immunizations, we observed no differences in T cell recovery in CD11c/A^b vs I-A^b+/- mice, even though CD11c/A^b mice lack endogenous MHCII-restricted CD4⁺ T cells and I-A^b+/- mice have full T cell compartments. Our results suggest that during the primary response, DC cognate interactions with CD4 T cells must regulate clonal competition for Ag and expansion. Similarly, our results indicate that the contraction phase of the response does not require cognate interactions with B cells, macrophages, or parenchymal cells. If TCR-mediated signaling is required for apoptosis of activated precursors in vivo, DC Ag presentation can mediate these events. Others have shown that DC Ag presentation is capable of mediating naive CD4⁺ T cell homeostasis (4) and our results extend those findings to the generation, maintenance, and contraction of effector CD4⁺ T cell numbers.

So what is the role for MHCII Ag presentation by non-DCs? Clearly, MHCII expression by B cells is required for isotype-switched Ab responses. In vitro experiments suggested that MHCII-negative B cells could receive help from activated CD4⁺ T cells (52). However, Williams et al. (14) found that MHCII on B cells was required for IgG production of T-dependent Ags in vivo, even in the context of productive T cell priming. Similarly, using a chimeric approach, Fillatreau and Gray (45) demonstrated that, although CD4⁺ T cell migration into B cell zones is independent of MHCII on B cells, isotype switching in this setting is defective. Our results agree with these in vivo observations, as DC MHCII expression cannot overcome the requirement for B cell-T cell cognate interactions. However, this is not to say that B cell Ag presentation cannot affect the T cell response, since a role for B cells in Th2 differentiation, CD4⁺ T cell memory responses, and peripheral tolerance has been previously reported (12, 13, 49, 50).

Finally, is there a role for macrophage and parenchymal MHCII expression? The roles of MHCII presentation could be more important during the effector phase of the response rather than Th1 polarization. We are currently addressing the sufficiency of DC MHCII Ag presentation in the effector phases of the T cell response. However, it has been suggested that Ag presentation by human endothelial cells facilitates the migration of Ag-specific cells into the tissues (19). Our findings and others indicate that neither Ag (20) nor MHCII expression by parenchymal cells is required for the migration of Ag-specific murine CD4⁺ T cells into peripheral sites. However, parenchymal MHCII presentation might play a role in the long-term maintenance of Ag-specific lymphocytes at sites of infection, which was not addressed by the current studies. Further study of the CD11c/A^b mouse model will allow us to delineate the distinct roles of different APCs in regulating Ag presentation to CD4⁺ T cells during immunity and autoimmunity.

	Acknowledgments

 
We thank Dr. Laurie Glimcher for supporting the initial phases of this work. We thank Chris Hunter, Steven Eck, and Susan Harless for constructive comments and critical review of this manuscript. We also thank Brian Busser, Avinash Bhandoola, Andrew Wells, Stefania Gallucci, and Larry Turka for helpful discussions and Traci Lifsted for expert technical advise with genotyping and breedings. Special thanks to the Flow Cytometry/Cell Sorting Facility and the Stem Cell Automacs Core at the University of Pennsylvania.

	Footnotes

 
¹ This work was supported by a Grant-in-Aid from the Pennsylvania/Delaware affiliate of the American Heart Association (to T.M.L.).

² Current address: Pharmacia, 4901 Searle Parkway, Stokie, IL, 60077.

³ Current address: Digital Gene Technologies, 11149 North Torrey Pines Road, La Jolla, CA 92037.

⁴ Address correspondence and reprint requests to Dr. Terri M. Laufer, Department of Medicine, University of Pennsylvania, 753 BRB II/III 421 Curie Boulevard, Philadelphia, PA 19104. E-mail address: tlaufer{at}mail.med.upenn.edu

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, mean fluorescence intensity; LN, lymph node.

Received for publication June 17, 2003. Accepted for publication September 15, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Cosgrove, D., D. Gray, A. Dierich, J. Kaufman, M. Lemeur, C. Benoist, D. Mathis. 1991. Mice lacking MHC class II molecules. Cell 66:1051.[Medline]
2. Grusby, M. J., R. S. Johnson, V. E. Papaioannou, L. H. Glimcher. 1991. Depletion of CD4⁺ T cells in major histocompatibility complex class II-deficient mice. Science 253:1417.[Abstract/Free Full Text]
3. Fehling, H. J., S. Viville, W. van Ewijk, C. Benoist, D. Mathis. 1989. Fine-tuning of MHC class II gene expression in defined microenvironments. Trends Genet. 5:342.[Medline]
4. Brocker, T.. 1997. Survival of mature CD4 T lymphocytes is dependent on major histocompatibility complex class II-expressing dendritic cells. J. Exp. Med. 186:1223.[Abstract/Free Full Text]
5. Guermonprez, P., J. Valladeau, L. Zitvogel, C. Thery, S. Amigorena. 2002. Antigen presentation and T cell stimulation by dendritic cells. Annu. Rev. Immunol. 20:621.[Medline]
6. Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y. J. Liu, B. Pulendran, K. Palucka. 2000. Immunobiology of dendritic cells. Annu. Rev. Immunol. 18:767.[Medline]
7. Maldonado-Lopez, R., M. Moser. 2001. Dendritic cell subsets and the regulation of Th1/Th2 responses. Semin. Immunol. 13:275.[Medline]
8. Boonstra, A., C. Asselin-Paturel, M. Gilliet, C. Crain, G. Trinchieri, Y. J. Liu, A. O’Garra. 2003. Flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing T helper type 1 and 2 cell development: dependency on antigen dose and differential toll-like receptor ligation. J. Exp. Med. 197:101.[Abstract/Free Full Text]
9. Krug, A., R. Veeraswamy, A. Pekosz, O. Kanagawa, E. Unanue, M. Colonna, M. Cella. 2003. Interferon-producing cells fail to induce proliferation of naive T cells but can promote expansion and T helper 1 differentiation of antigen-experienced unpolarized T cells. J. Exp. Med. 197:899.[Abstract/Free Full Text]
10. De Smedt, T., B. Pajak, G. G. Klaus, R. J. Noelle, J. Urbain, O. Leo, M. Moser. 1998. Antigen-specific T lymphocytes regulate lipopolysaccharide-induced apoptosis of dendritic cells in vivo. J. Immunol. 161:4476.[Abstract/Free Full Text]
11. Constant, S. L.. 1999. B lymphocytes as antigen-presenting cells for CD4⁺ T cell priming in vivo. J. Immunol. 162:5695.[Abstract/Free Full Text]
12. Linton, P. J., J. Harbertson, L. M. Bradley. 2000. A critical role for B cells in the development of memory CD4 cells. J. Immunol. 165:5558.[Abstract/Free Full Text]
13. van Essen, D., P. Dullforce, T. Brocker, D. Gray. 2000. Cellular interactions involved in Th cell memory. J. Immunol. 165:3640.[Abstract/Free Full Text]
14. Williams, G. S., A. Oxenius, H. Hengartner, C. Benoist, D. Mathis. 1998. CD4⁺ T cell responses in mice lacking MHC class II molecules specifically on B cells. Eur. J. Immunol. 28:3763.[Medline]
15. DiMolfetto, L., H. A. Neal, A. Wu, C. Reilly, D. Lo. 1998. The density of the class II MHC T cell receptor ligand influences IFN-/IL-4 ratios in immune responses in vivo. Cell. Immunol. 183:70.[Medline]
16. Yamane, H., T. Kato, H. Nariuchi. 1999. Effective stimulation for IL-12 p35 mRNA accumulation and bioactive IL-12 production of antigen-presenting cells interacted with Th cells. J. Immunol. 162:6433.[Abstract/Free Full Text]
17. Bright, J. J., Z. Xin, S. Sriram. 1999. Superantigens augment antigen-specific Th1 responses by inducing IL-12 production in macrophages. J. Leukocyte Biol. 65:665.[Abstract]
18. Trinchieri, G.. 2003. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat. Rev. Immunol.. 3:133.[Medline]
19. Marelli-Berg, F. M., L. Frasca, L. Weng, G. Lombardi, R. I. Lechler. 1999. Antigen recognition influences transendothelial migration of CD4⁺ T cells. J. Immunol. 162:696.[Abstract/Free Full Text]
20. Reinhardt, R. L., D. Bullard, C. Weaver, M. Jenkins. 2003. Preferential accumulation of antigen-specific effector CD4 T cells at an antigen injection site involves CD62E-dependent migration but not local proliferation. J. Exp. Med. 197:751.[Abstract/Free Full Text]
21. Edwards, A., A. C. Reis de Sousa. 2003. Toll like receptor expression in murine Dc subsets: lack of TLR7 expression by CD8+ DC correlates with unresponsiveness to imidazoquinoles. Eur. J. Immunol. :827.
22. Choi, E., K. McIntyre, R. N. Germain, J. G. Seidman. 1983. Murine I-A chain polymorphism: nucleotide sequences of three allelic I-A genes. Science 221:283.[Abstract/Free Full Text]
23. Mombaerts, P., A. R. Clarke, M. A. Rudnicki, J. Iacomini, S. Itohara, J. J. Lafaille, L. Wang, Y. Ichikawa, R. Jaenisch, M. L. Hooper, et al 1992. Mutations in T-cell antigen receptor genes and block thymocyte development at different stages. Nature 360:225.[Medline]
24. Grubin, C. E., S. Kovats, P. deRoos, A. Y. Rudensky. 1997. Deficient positive selection of CD4 T cells in mice displaying altered repertoires of MHC class II-bound self-peptides. Immunity 7:197.[Medline]
25. Barnden, M. J., J. Allison, W. R. Heath, F. R. Carbone. 1998. Defective TCR expression in transgenic mice constructed using cDNA-based - and -chain genes under the control of heterologous regulatory elements. Immunol. Cell Biol. 76:34.[Medline]
26. Larsen, C. P., R. M. Steinman, M. Witmer-Pack, D. F. Hankins, P. J. Morris, J. M. Austyn. 1990. Migration and maturation of Langerhans cells in skin transplants and explants. J. Exp. Med. 172:1483.[Abstract/Free Full Text]
27. Belkaid, Y., H. Jouin, G. Milon. 1996. A method to recover, enumerate and identify lymphomyeloid cells present in an inflammatory dermal site: a study in laboratory mice. J. Immunol. Methods 199:5.[Medline]
28. Inaba, K., M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. Muramatsu, R. M. Steinman. 1992. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 176:1693.[Abstract/Free Full Text]
29. Wells, A. D., H. Gudmundsdottir, L. A. Turka. 1997. Following the fate of individual T cells throughout activation and clonal expansion: signals from T cell receptor and CD28 differentially regulate the induction and duration of a proliferative response. J. Clin. Invest. 100:3173.[Medline]
30. Gudmundsdottir, H., A. D. Wells, L. A. Turka. 1999. Dynamics and requirements of T cell clonal expansion in vivo at the single-cell level: effector function is linked to proliferative capacity. J. Immunol. 162:5212.[Abstract/Free Full Text]
31. Kearney, E. R., T. L. Walunas, R. W. Karr, P. A. Morton, D. Y. Loh, J. A. Bluestone, M. K. Jenkins. 1995. Antigen-dependent clonal expansion of a trace population of antigen-specific CD4⁺ T cells in vivo is dependent on CD28 costimulation and inhibited by CTLA-4. J. Immunol. 155:1032.[Abstract]
32. Vezys, V., A. L. Marzo, L. Lefrancois. 2001. Tissue-level regulation of Th1 and Th2 primary and memory CD4 T cells in response to Listeria infection. Science 291:2413.[Abstract/Free Full Text]
33. Garside, P., E. Ingulli, R. R. Merica, J. G. Johnson, R. J. Noelle, M. K. Jenkins. 1998. Visualization of specific B and T lymphocyte interactions in the lymph node. Science 281:96.[Abstract/Free Full Text]
34. Crowley, M. T., C. R. Reilly, D. Lo. 1999. Influence of lymphocytes on the presence and organization of dendritic cell subsets in the spleen. J. Immunol. 163:4894.[Abstract/Free Full Text]
35. Jung, S., D. Unutmaz, P. Wong, G. Sano, K. De los Santos, T. Sparwasser, S. Wu, S. Vuthoori, K. Ko, F. Zavala, et al 2002. In vivo depletion of CD11c⁺ dendritic cells abrogates priming of CD8⁺ T cells by exogenous cell-associated antigens. Immunity 17:211.[Medline]
36. Brocker, T., M. Riedinger, K. Karjalainen. 1997. Targeted expression of major histocompatibility complex (MHC) class II molecules demonstrates that dendritic cells can induce negative but not positive selection of thymocytes in vivo. J. Exp. Med. 185:541.[Abstract/Free Full Text]
37. Ngo, V. N., R. J. Cornall, J. G. Cyster. 2001. Splenic T zone development is B cell dependent. J. Exp. Med. 194:1649.[Abstract/Free Full Text]
38. O’Connell, P. J., A. E. Morelli, A. J. Logar, A. W. Thomson. 2000. Phenotypic and functional characterization of mouse hepatic CD8+ lymphoid-related dendritic cells. J. Immunol. 165:795.[Abstract/Free Full Text]
39. Byersdorfer, C. A., D. D. Chaplin. 2001. Visualization of early APC/T cell interactions in the mouse lung following intranasal challenge. J. Immunol. 167:6756.[Abstract/Free Full Text]
40. Julia, V., E. M. Hessel, L. Malherbe, N. Glaichenhaus, A. O’Garra, R. L. Coffman. 2002. A restricted subset of dendritic cells captures airborne antigens and remains able to activate specific T cells long after antigen exposure. Immunity 16:271.[Medline]
41. Ernst, B., D. S. Lee, J. M. Chang, J. Sprent, C. D. Surh. 1999. The peptide ligands mediating positive selection in the thymus control T cell survival and homeostatic proliferation in the periphery. Immunity 11:173.[Medline]
42. Bird, J. J., D. R. Brown, A. C. Mullen, N. H. Moskowitz, M. A. Mahowald, J. R. Sider, T. F. Gajewski, C. R. Wang, S. L. Reiner. 1998. Helper T cell differentiation is controlled by the cell cycle. Immunity 9:229.[Medline]
43. Reinhardt, R. L., A. Khoruts, R. Merica, T. Zell, M. K. Jenkins. 2001. Visualizing the generation of memory CD4 T cells in the whole body. Nature 410:101.[Medline]
44. Constant, S., D. Sant’Angelo, T. Pasqualini, T. Taylor, D. Levin, R. Flavell, K. Bottomly. 1995. Peptide and protein antigens require distinct antigen-presenting cell subsets for the priming of CD4⁺ T cells. J. Immunol. 154:4915.[Abstract]
45. Fillatreau, S., D. Gray. 2003. T cell accumulation in B cell follicles is regulated by dendritic cells and is independent of B cell activation. J. Exp. Med. 197:195.[Abstract/Free Full Text]
46. Josien, R., H. L. Li, E. Ingulli, S. Sarma, B. R. Wong, M. Vologodskaia, R. M. Steinman, Y. Choi. 2000. TRANCE, a tumor necrosis factor family member, enhances the longevity and adjuvant properties of dendritic cells in vivo. J. Exp. Med. 191:495.[Abstract/Free Full Text]
47. von Stebut, E., Y. Belkaid, B. V. Nguyen, M. Cushing, D. L. Sacks, M. C. Udey. 2000. Leishmania major-infected murine Langerhans cell-like dendritic cells from susceptible mice release IL-12 after infection and vaccinate against experimental cutaneous leishmaniasis. Eur. J. Immunol. 30:3498.[Medline]
48. Attinger, A., H. R. MacDonald, H. Acha-Orbea. 2001. Lymphoid environment limits superantigen and antigen-induced T cell proliferation at high precursor frequency. Eur. J. Immunol. 31:884.[Medline]
49. Harbertson, J., E. Biederman, Y. Zhang, S. M. Bradley, P. J. Linton, L. M. Bradley. 2002. Availability of antigen-presenting cells can determine the extent of CD4 effector expansion and priming for secretion of Th2 cytokines in vivo. Eur. J. Immunol. 32:2338.[Medline]
50. Linton, P., B. Bautista, E. Biederman, E. Bradley, J. Harbertson, R. Kondrack, R. Padrick, L. Bradley. 2003. Costimulation via OX40L expressed by B cells is sufficient to determine the extent of primary CD4 cell expansion and Th2 cytokine secretion in vivo. J. Exp. Med. 197:875.[Abstract/Free Full Text]
51. Campbell, D. J., B. C. Butcher. 2002. Rapid acquisition of tissue-specific homing phenotypes by CD4⁺ T cells activated in cutaneous or mucosal lymphoid tissues. J. Exp. Med. 195:135.[Abstract/Free Full Text]
52. Markowitz, J. S., P. R. Rogers, M. J. Grusby, D. C. Parker, L. H. Glimcher. 1993. B lymphocyte development and activation independent of MHC class II expression. J. Immunol. 150:1223.[Abstract]
53. Busser, B. W., B. S. Adair, J. Erikson, and T. M. Laufer. Loss of anti-dsDNA B cell tolerance requires activation of a diverse repertoire of autoreactive T cells. J. Clin. Invest. In press.

This article has been cited by other articles:

				T. M Strutt, J. Uzonna, K. K McKinstry, and P. A Bretscher Activation of thymic T cells by MHC alloantigen requires syngeneic, activated CD4+ T cells and B cells as APC Int. Immunol., May 1, 2006; 18(5): 719 - 728. [Abstract] [Full Text] [PDF]

				M. P. Lemos, F. Esquivel, P. Scott, and T. M. Laufer MHC Class II Expression Restricted to CD8{alpha}+ and CD11b+ Dendritic Cells Is Sufficient for Control of Leishmania major J. Exp. Med., March 1, 2004; 199(5): 725 - 730. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) A correction has been published Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles