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

This Article Abstract Full Text (PDF) 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 Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cook, L. Articles by Born, W. K. Search for Related Content PubMed PubMed Citation Articles by Cook, L. Articles by Born, W. K.

Evidence That CD8⁺ Dendritic Cells Enable the Development of T Cells That Modulate Airway Hyperresponsiveness¹

Laura Cook^*, Nobuaki Miyahara, Niyun Jin^*, J. M. Wands^*, Christian Taube, Christina L. Roark^*, Terry A. Potter^*, Erwin W. Gelfand, Rebecca L. O'Brien^* and Willi K. Born^2,*

^* Department of Immunology and Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Airway hyperresponsiveness (AHR), a hallmark of asthma and several other diseases, can be modulated by T cells. In mice sensitized and challenged with OVA, AHR depends on allergen-specific β T cells; but V1⁺ T cells spontaneously enhance AHR, whereas V4⁺ T cells, after being induced by airway challenge, suppress AHR. The activity of these T cell modulators is allergen nonspecific, and how they develop is unclear. We now show that CD8 is essential for the development of both the AHR suppressor and enhancer T cells, although neither type needs to express CD8 itself. Both cell types encounter CD8-expressing non-T cells in the spleen, and their functional development in an otherwise CD8-negative environment can be restored with transferred spleen cell preparations containing CD8⁺ dendritic cells (DCs), but not CD8⁺ T cells or CD8^– DCs. Our findings suggest that CD8⁺ DCs in the lymphoid tissues enable an early step in the development of T cells through direct cell contact. DC-expressed CD8 might take part in this interaction.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Airway hyperresponsiveness (AHR)³ is a symptom in several potentially life-threatening diseases, including asthma and chronic obstructive pulmonary disease. The pathways leading to AHR are not yet completely mapped. In allergic asthma, Ag-specific T cells are initiators of a cascade of immune-dependent mechanisms that result in the production of the critical cytokine IL-13, eosinophilic airway inflammation, allergen-specific IgE Abs, mast cell activation, goblet cell differentiation and mucus production, and AHR (1, 2). The development and role of allergen-specific β T cells have been studied extensively in animal models of allergic airway inflammation and AHR. In the process, it became apparent that innate Ag-nonspecific T cells, which are capable of suppressing and enhancing AHR in allergic airway disease and other pathological conditions of the lung (3, 4, 5, 6, 7), were involved as well. Both β T cells and T cells are included among these innate AHR-modulating T cells and are potentially attractive targets for immune intervention (8). However, little is known about these cells and their development. We have studied AHR-modulating T cells in vivo. In mice sensitized and challenged with OVA that display hypersensitivity when given the cholinergic agonist methacholine (MCh) (9), we found that T cells can dramatically alter AHR whereas their effect on airway inflammation is comparatively small (5). The T cells capable of modulating AHR belong to two subsets distinguished by their TCRs: a V1⁺ subset that contains T cells spontaneously capable of enhancing AHR (10, 11) and a V4⁺ subset that contains T cells that can be induced to suppress AHR (12, 13, 14, 15). Neither cell type requires specific Ag priming (11, 15), and it is unclear how these AHR modulators develop. However, both are found in spleen and lung (16). Moreover, following airway challenge, selectively depleting either subset in the lung (using aerosolized, inhaled anti-TCR Abs) alters AHR in the predicted fashion, i.e., depletion of V1⁺ cells decreases and depletion of V4⁺ cell increases AHR (8, 12), suggesting that the T cells within the challenged lung are functionally competent.

In the development of Ag-specific T cells, dendritic cells (DCs) are critical (17). The role of DCs in the development of innate T cells is less well defined. Evidence that T cells and DCs interact has come mainly from studies in vitro. These studies revealed maturational effects on both the DCs and the T cells (18, 19, 20, 21, 22, 23, 24), raising the question of whether such interactions might also be critical for the functional development of T cells in vivo. However, DCs are heterogeneous and their functions are diverse (25, 26, 27, 28, 29, 30, 31). The present study began with the finding that AHR suppression by T cells could not be demonstrated in mice genetically deficient in CD8 and with our attempts to determine which cells express the required CD8. Using adoptive cell transfer, we find that the T cells themselves need not express CD8, but that CD8 is required in the donor mice in which the T cells develop. Further, the development of T cells capable of AHR suppression can be restored in CD8-deficient mice by reconstituting them with cell preparations containing CD8⁺ DCs, but not with CD8-expressing T cells or CD8^– DCs. Mice deficient in CD8 also fail to give rise to T cells capable of AHR enhancement, and again their development can be restored by transfer of CD8⁺ DCs, suggesting that interactions with CD8⁺ DCs in lymphoid tissues represent a critical step in the development of T cells toward functional competence.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Animals

C57BL/6 (B6), B6.CD8^–/–, B6.TCR-β^–/–, B6.TCR-^–/–, and B6.TCR-β^–/–/^–/– mice were purchased from The Jackson Laboratory; TCR-V4/6^–/– mice deficient in V4⁺ and V6⁺ T cells (32) were a gift from K. Ikuta (Kyoto University, Kyoto, Japan). They were backcrossed to the B6 genetic background and established after 11 backcrossed generations (designated B6.TCR-V4/6^–/–). B6.TCR-V4/6^–/–CD8^–/– mice were generated starting from the F2 generation of a cross between B6.CD8^–/– and B6.TCR-V4/6^–/– mice. All mice were maintained on an OVA-free diet and were cared for at the National Jewish Medical and Research Center (Denver, Colorado), following guidelines for immune deficient animals when necessary. All experiments were conducted under a protocol approved by the Institutional Animal Care and Use Committee of the National Jewish Medical and Research Center.

Sensitization and airway challenge

Mice received the following treatments: no OVA treatment; sensitization to OVA by i.p. injection of 20 µg of OVA (grade V; Sigma-Aldrich) emulsified in 2.25 mg of alum (AlumImject; Pierce) in a total volume of 100 µl on days 0 and 14 (2ip); or sensitization followed by airway exposure to nebulized OVA (1% in saline) using ultrasonic nebulization (particle size 3–5 µm) for 20 min on days 28, 29, and 30 (2ip3N). Donors of DC preparations either received no treatment or only the sensitizing treatment (2ip). Airway responsiveness was assessed 48 h after the last nebulized OVA exposure for 2ip3N-treated mice.

Depletion with mAb against TCR

Cell depletion was achieved by injection into the tail vein of 200 µg of hamster mAb against V4 (clone UC3) (33) 3 days before the first OVA challenge, and depletion was assessed as described (10). Sham depletion was conducted using hamster Ig (The Jackson Laboratory). Throughout this article, we use the nomenclature for murine TCR-V genes introduced by Heilig and Tonegawa (34).

Determination of airway responsiveness

Airway responsiveness was assessed as a change in respiratory system resistance after provocation with aerosolized MCh using a method previously described in detail (5). MCh aerosol was administered for 10 s (60 breaths/min, 0.5 ml of tidal volume) in increasing concentrations as indicated in the figures. Maximum values of lung resistance and minimum values of dynamic compliance were recorded and expressed as percentage change from baseline after saline aerosol.

Histological analysis of the spleen

For tissue preparation, sectioning, and staining, essentially the same methods were used as those described previously for the analysis of T cells in the lung (16). Briefly, sections of 5- to 10-µm thickness cut from frozen blocks were mounted on microscopic slides, air dried, dehydrated in acetone, hydrated, blocked with a mixture of normal mouse serum and Avidin, washed, stained with primary and secondary Abs, and finally mounted with coverslips. Slides were viewed using a Leica DMRXMA upright fluorescent microscope, and digital images were generated on an Apple MacIntosh computer connected with the microscope, using the SlideBook imaging program (3I Inc.). The tissue autofluorescence (green) was used to distinguish overall tissue organization, especially the localization of central arterioles and the density of the cellular distribution. The higher cell density of the white pulp around the arterioles was used to determine the extent of the periarteriolar lymphoid sheath (PALS). Cells and cell contacts were enumerated and compared either across the entire tissue (whole spleen) or limited to the PALS. Abs used for histology were anti-TCR- (mAb GL3), anti-V4 (mAb UC3), anti-V1 (mAb 2.11), anti-CD8 (mAb 53-6.7; eBioscience), anti-CD11c (mAb HL3; BD Pharmingen), anti-CD103 (mAb 2E7, eBisocience), and anti-DEC-205 (American Type Culture Collection mAb no. HB 290) plus secondary reagents as described (16).

Cell preparation from spleen and T cell sorting using the MoFlo cell sorter

A suspension of splenocytes was prepared by mechanical dispersion. Suspended cells were treated with Gey’s red cell lysis solution. Nylon wool-nonadherent cells were prepared from spleens of OVA-sensitized and challenged B6.TCR-β^–/– mice 2 days after the last challenge. These preparations contained >75% T cells. Total cell counts were determined using a Coulter counter. Nylon wool-nonadherent cells in PBS with 5% FBS were incubated with FITC-conjugated anti-V4 mAb (clone UC3) and PE-conjugated anti-CD8 (clone 53-6.7; BD Pharmingen) or PE-conjugated anti-CD8β (53-5.8; BD Pharmingen) (20 min at 4°C), and then washed. Cells were next sorted based on their expression of V4 and CD8 ( or β) using a MoFlo cell sorter. Purified V4⁺/CD8⁺ or V4⁺/CD8^– cells were washed in PBS and resuspended at 5 x 10⁴ cells/ml PBS, and 1 x 10⁴ cells/mouse were injected in 200 µl of PBS via the tail vein into OVA-sensitized B6.TCR-V4/6^–/– mice within 1 h before the first airway challenge.

Adoptive transfer of T cells

Nylon wool-nonadherent cells were prepared from spleens of OVA-sensitized and challenged B6.TCR-β^–/– or B6.CD8^–/– mice 2 days after the last challenge. These cells contained >75% T cells. Total cell counts were determined using a Coulter counter. Nylon wool-nonadherent cells in PBS with 5% FBS were incubated with biotinylated anti-V4 mAb (clone UC3) or anti-V1 mAb (clone 2.11) (35) (20 min at 4°C) and then washed and incubated with streptavidin-conjugated magnetic beads (streptavidin microbeads; Miltenyi Biotec) for 15 min at 4°C and passed twice through magnetic columns to purify V4⁺ or V1⁺ cells. This produced a cell population containing >80% V4⁺ or V1⁺ viable cells as determined by two-color staining with anti-TCR- and anti-V4 or anti-V1 mAbs. These splenic V4⁺ or V1⁺ cells were washed in PBS and resuspended to 5 x 10⁴ cells/ml PBS and 1 x 10⁴ cells/mouse were injected in 200 µl of PBS via the tail vein into OVA-sensitized B6.TCR-V4/6^–/– or B6.TCR-^–/– mice within 1 h before the first airway challenge.

Phenotype of splenocytes and enriched DC from B6.TCR-β^–/–/^–/– mice

Red cell-depleted splenocytes from naive or 2ip-treated B6.TCR-β^–/–/^–/– mice (or magnetically enriched DCs) were incubated with allophycocyanin-conjugated anti-CD8 mAb (clone 53-6.7, eBioscience), FITC conjugated anti-CD11c mAb (clone HL3, Pharmingen), biotinylated anti-CD45R (B220, clone RA3-6B2; eBioscience), biotinylated anti-CD3 mAb (clone KT3), and PE-conjugated anti-MHC II (clone M5/114.15.2; BD Pharmingen) in PBS with 5% FBS (20 min at 4°C) and then washed and analyzed on a FACScan flow cytometer.

Adoptive transfer of accessory non-T cells

Cells were isolated from the spleen of untreated or 2ip-treated B6.TCR-β^–/–/^–/– mice and were treated with Gey’s red cell lysis solution. To reconstitute with whole splenocytes, one "spleen equivalent" (5 x 10⁷ cells) was then transferred per 2ip-treated B6.CD8^–/– mouse. A small sample of these cells was analyzed for CD8 expression by flow cytometry (mAb 53-6.7-PE; BD Pharmingen). Approximately 9% were found to be CD8⁺. Reconstituted B6.CD8^–/– mice were then nebulized as described earlier and used as donors of V4⁺ or V1⁺ cells. To reconstitute with CD8-enriched or CD8-depleted splenocytes, the red cell depleted splenocytes from 2ip-treated B6.TCR-β^–/–/^–/– or B6.TCR-^–/– mice (for CD8⁺ T cell adoptive transfer) were incubated with biotinylated anti-CD8 mAb 53-6.7 in PBS with 5% FBS (20 min at 4°C) and then washed and incubated with streptavidin-conjugated magnetic beads (streptavidin microbeads; Miltenyi Biotec) for 15 min at 4°C and passed twice over MS magnetic columns to purify CD8⁺ cells. This produced a CD8-enriched fraction containing 45% CD8⁺ cells as determined by flow cytometry and a CD8-depleted fraction containing <1% CD8⁺ cells. Approximately 1 x 10⁶ viable enriched cells were transferred to 2ip-treated B6.CD8^–/– recipients. The B6.CD8^–/– mouse was then nebulized as described earlier and these mice were used as donors of V4⁺ cells. Finally, to reconstitute with more highly enriched CD8⁺ DCs, a CD8⁺ purification kit (Miltenyi Biotec) was used as per the manufacturer’s recommendations. Briefly, freshly isolated, red cell-depleted splenocytes derived from the B6.TCR-β^–/–/^–/– mice were incubated with a biotinylated Ab mixture (anti-CD90, anti-CD45R, and anti-CD49b) in PBS with 5% FBS (10 min on ice), incubated with anti-biotin microbeads (15 min, on ice), and then washed and passed over a LD magnetic column to enrich DCs. The enriched DCs were incubated with anti-CD8 (clone Ly-2) microbeads (30 min on ice) and then washed and passed twice over a MS magnetic column retaining the CD8⁺ DCs. In the worst case (with 2ip-treated mice), this still produced a preparation containing 60–70% viable CD8⁺ DCs based on the dual criterion of MHC class II and CD11c expression. The concentration of CD8⁺ DC in this preparation might actually be higher due to variable MHC class II expression. A total of 1.3 x 10⁵ of these enriched cells containing 10⁵ CD8⁺ DCs were transferred to 2ip-treated B6.CD8^–/– recipients, which were then nebulized as described above and used as donors of V4⁺ cells. Donors of V1⁺ cells (nonsensitized B6.CD8^–/– mice that received enriched CD8⁺ DC from nonsensitized B6.TCR-β^–/–/^–/– mice; these could be obtained at a higher purity than those from 2ip-treated mice) remained nonchallenged.

Statistical analysis

Data are presented as means ± SEM. The unpaired t test was used for two group comparisons and ANOVA was used for analysis of differences in three or more groups. Pairwise comparisons were performed using the Tukey-Kramer honest significant difference test. Statistically significant levels are indicated as follows: one symbol (*, #, or +) = p < 0.05; two symbols = p < 0.01; three symbols = p < 0.001.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
AHR-suppressive T cells depend on the CD8 molecule but need not themselves express CD8

We have previously found that V4⁺ T cells suppress AHR in several different mouse strains, including B6 (12, 13, 14, 15). In this study we have used B6 mice to take advantage of available mutations on this background. As predicted by earlier studies (36), in comparison with wild-type mice (Fig. 1a), mice deficient in CD8 (B6.CD8^–/–) (Fig. 1b) exhibited much-reduced AHR after being sensitized and challenged with OVA. Unexpectedly, however, i.v. injection of a cell-depleting anti-TCR-V4 Ab (mAb UC3) that increases AHR in wild-type B6 mice (Fig. 1a) failed to increase AHR in the CD8-deficient mice (Fig. 1b). This difference suggested a role for CD8 in the suppression of AHR by T cells, but it remained unclear when the molecule might be needed and which cells must express it. To address these questions, we chose a cell transfer approach. We compared mice adoptively transferred with purified V4⁺ T cells derived from the spleens of OVA-sensitized and -challenged wild-type B6 (Fig. 1c) or B6.CD8^–/– donors (Fig. 1d) by using recipients deficient in V4⁺ cells but expressing normal levels of CD8 (B6.TCR-V4/6^–/–). These B6.TCR-V4/6^–/– mice develop strong AHR following OVA sensitization and challenge (13). After cell transfer, the B6.TCR-V4/6^–/– mice that received V4⁺ cells from wild-type-mice exhibited reduced AHR (Fig. 1c), but those that received V4⁺ cells from B6.CD8^–/– mice did not (Fig. 1d). In a similar comparative experiment transferring T cells derived from the lung, again the wild type-derived cells suppressed AHR whereas the cells from CD8-deficient mice failed to do so (not shown). These results indicated that CD8 expression in the cell-transfer recipients is not sufficient to support the AHR modulating function of the T cells. CD8 expression by the donor cells is required, either by the T cells themselves or by other cells in the donors that might determine the development of the T cells. Whether CD8 might in addition play a role during T cell-mediated AHR suppression in the cell transfer recipients was not pursued because the CD8-deficient recipient mice bred for this purpose (B6.TCR-V4/6^–/–CD8^–/–) failed to develop AHR (not shown).

View larger version (34K): [in this window] [in a new window]   FIGURE 1. AHR in mice after systemic sensitization and airway challenge with OVA (2ip3N protocol). Lung resistance (RL) and dynamic compliance (Cdyn) as percentage changes from saline controls in relation to increasing doses of aerosolized MCh are shown. Treatments were as follows: none (NT), i.p. sensitization and airway challenge (2ip3N), 2ip3N plus hamster IgG i.v. (2ip3N + HIgG), 2ip3N plus anti-V4 mAb UC3 i.v. (2ip3N + UC3), 2ip3N plus purified V4+ cells from 2ip3N-treated donors i.v., wild type (2ip3N + wt V4+ cells) or CD8–/– (2ip3N + CD8– V4+ cells). a, Normal B6 mice after 2ip3N or 2ip3N plus depletion of V4+ cells with mAb UC3 (*, nontreated compared with 2ip3N; #, 2ip3N compared with 2ip3N plus UC3). b, B6.CD8–/– mice after 2ip3N plus depletion of V4+ cells with mAb UC3 (*, nontreated compared with 2ip3N). c, B6.TCR-V4/6–/– mice after 2ip3N or 2ip3N plus V4+ cells from B6.TCR-β–/– (CD8 wild type) donors (*, nontreated compared with 2ip3N; there was no significant difference between nontreated and 2ip3N plus WT V4 cells). d, B6.TCR-V4/6–/– mice after 2ip3N or 2ip3N plus V4+ cells from B6.CD8–/– donors (there was no significant difference between 2ip3N and 2ip3N plus CD8–/– V4 cells at any dose of MCh). One symbol (* or #) = p < 0.05; two symbols = p < 0.01; three symbols = p < 0.001.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. AHR in T cell-deficient recipients reconstituted with CD8-selected V4+ cells after systemic sensitization and airway challenge with OVA (2ip3N protocol). Lung resistance (RL) and dynamic compliance (Cdyn) as percentage changes from saline controls in relation to increasing doses of aerosolized MCh are shown. Recipients are B6.TCR-V4/6–/– mice and donors are 2ip3N-treated B6.TCR-β–/– or B6 mice. Treatments are as follows: none (NT), systemic sensitization and airway challenge (2ip3N), 2ip3N plus selected purified V4+/CD8β+ (2ip3N + CD8β+ V4 cells), V4+/CD8β– (2ip3N + CD8β– V4 cells), V4+/CD8+ (2ip3N + CD8+ V4 cells), or V4+/CD8– cells (2ip3N + CD8– V4 cells) from the donors. a, Recipients untreated or treated with 2ip3N or 2ip3N plus selected V4+/CD8β+ or V4+/CD8β– cells from B6.TCR-β–/– donors (*, nontreated compared with 2ip3N). b, Recipients untreated or treated with 2ip3N or 2ip3N plus selected V4+/CD8+ or V4+/CD8– cells from B6 donors (*, nontreated compared with 2ip3N; #, 2ip3N compared with 2ip3N plus CD8+ V4 cells; +, 2ip3N compared with 2ip3N plus CD8– V4 cells). One symbol (*, #, or +) = p < 0.05; two symbols = p < 0.01.

Encounters between T cells and CD8⁺ non-T cells in the spleen

Because T cells prepared from the spleen are capable of modulating AHR, we examined their distribution and cell contacts. In B6 (Fig. 3a) and B6.TCR-β^–/– mice (Fig. 3b), both of which give rise to functionally normal AHR-regulatory T cells (11, 13), T cells were mainly present in the PALS, although some were also detected outside the PALS. The T cells were often in close proximity/contact with DEC-205⁺ or CD8⁺ cells (Fig. 3, a and b). The CD8⁺ cells are not β T cells, because they are present in the spleen of B6.TCR-β^–/– mice. Both V4⁺ and V1⁺ cells were found in such interactions (Fig. 3, c and d) with little change following sensitization and challenge (Fig. 4). The frequency of CD8⁺ non-T cells in contact with T cells was higher inside the PALS (50–60%) than in the spleen in general (30–40%) (Fig. 4). However, the high frequency of such contacts might be a peculiarity of B6.TCR-β^–/– mice. In accordance with the phenotype of "conventional" CD8⁺ DCs (31), the CD8⁺ non-T cells in the PALS of the B6.TCR-β^–/– mice coexpressed CD11c (Fig. 3e), and many coexpressed CD103 and DEC-205 (Fig. 3, h and i). Collectively, these findings suggest that T cells, including those types implicated in the modulation of AHR, encounter CD8⁺ DCs in the spleen.

View larger version (133K): [in this window] [in a new window]   FIGURE 3. Localization of T cells, CD8+ DCs, and CD8– OVA/alum-containing phagocytes in the spleen. Frozen sections of spleen stained with mAbs, tissue autofluorescence (green), Ab staining (red or blue), or coincidence of Ab staining (pink/purple) are shown. a, B6 spleen stained with anti-TCR- mAb (red) and anti-DEC-205 mAb (blue). b, B6.TCR-β–/– spleen stained with anti-TCR- mAb (red) plus anti-CD8 mAb (blue). c and d, B6.TCR-β–/– spleen stained with anti-TCR-V4 mAb (red) plus anti-CD8 mAb (blue) (c) or with anti-TCR-V1 mAb (red) plus anti-CD8 mAb (blue) (d). e, B6.TCR-β–/– spleen stained with anti-CD11c mAb (red) plus anti-CD8 mAb (blue); cells coexpressing these markers appear violet. f, B6.CD8–/– spleen stained with anti-TCR- mAb (red). g, B6.TCR-β–/––/– spleen stained with anti-CD8 mAb. h, B6.TCR-β–/– spleen stained with an Ab against CD103 (red) plus anti-CD8 mAb (blue); cells coexpressing these markers appear violet. i, B6.TCR-β–/– spleen stained with an Ab against DEC-205 (red) plus anti-CD8 mAb 53-6/7 (blue); cells coexpressing these markers appear violet. j, B6.CD8–/– spleen stained with anti-TCR- mAb (red) plus anti-DEC-205 mAb (blue). k, 2ip-treated B6.CD8–/– spleen after transfer of CD8+ cells from the spleen of a 2ip-treated B6.TCR-β–/––/– mouse stained with anti-TCR- mAb (red) plus anti-CD8 mAb (blue); enlarged detail showing two endogenous T cells in contact with transferred CD8+ DCs. l, Same tissue and staining as in k; additional detail: OVA/alum appears as bright white/yellow fluorescence in CD8–TCR-– phagocytes, which are found in close proximity to both a T cell and a CD8+ DC.

View larger version (18K): [in this window] [in a new window]   FIGURE 4. Distribution of contacts between T cells and CD8+ DCs in the spleen. Frozen sections of spleen stained with mAbs were evaluated for relative frequencies of T cell-DC contacts. a, B6.TCR-β–/– spleen nontreated cells and 2ip3N-treated cell;, fractions of TCR-+, V1+, and V4+ cells in contact with CD8+ non-T cells. b, B6.TCR-β–/– spleen nontreated and 2ip3N-treated cells; comparison of PALS and "outside the PALS" fractions of TCR-+ cells in contact with CD8+ non-T cells. c, B6.TCR-β–/– spleen nontreated and 2ip3N-treated cells; fractions of CD8+ non-T cells in contact with TCR-+, V1+, and V4+ cells. d, B6.TCR-β–/– spleen nontreated and 2ip3N-treated cells; comparison of PALS and "outside the PALS" fractions of CD8+ non-T cells in contact with TCR-+ cells.

Enriched CD8⁺ DCs enable the development of AHR-suppressive T cells in mice lacking CD8

Because CD8-negative T cells were capable of suppressing AHR (Fig. 2) and because of our previous finding that the AHR suppressors can develop in mice lacking all β T cells (5, 12, 13), we concluded that CD8⁺ non-T cells must satisfy the CD8 requirement. Furthermore, the histological data of this study suggested CD8⁺ DCs as candidate CD8⁺ non-T cells. Therefore, we tested whether B6.CD8^–/– mice might be able to generate V4⁺ AHR suppressors if reconstituted with CD8⁺ DCs. To avoid all CD8⁺ T cells, we chose B6.TCR-β^–/–/^–/– mice as a source of the CD8⁺ accessory cells. Furthermore, because the AHR-suppressive V4⁺ T cells from the spleen require prior sensitization and challenge (15), we OVA sensitized (2ip) both the donors of the CD8⁺ accessory cells and the CD8^–recipients before i.v. transfer of the accessory cells. The reconstituted B6.CD8^–/– mice were then immediately challenged with OVA (3N) so that all cells in these mice were exposed to the full protocol of sensitization and challenge (2ip3N). V4⁺ T cells of these mice were subsequently tested for their ability to suppress AHR in B6.TCR-V4/6^–/– recipients, using the same method as described in Fig. 1. Indeed, these V4⁺ cells suppressed AHR, indicating that genetically CD8-deficient V4⁺ T cells still retain the potential of developing into AHR-suppressors and that B6.TCR-β^–/–/^–/– mice (CD8wt) contain non-T cells capable of restoring their development (Fig. 5a). To determine whether this capability was contained within the CD8⁺ non-T cells themselves, we compared CD8⁺ and CD8^– splenocytes from B6.TCR-β^–/–/^–/– mice for their ability to restore T cell-development. Only the CD8⁺ fraction enabled development of the AHR suppressors, suggesting that the reconstituting cells themselves must express CD8 (Fig. 5b). This experiment also ruled out the possibility that the mere process of cell transfer induced the AHR suppressors. Next, to better enrich splenic CD8⁺ DCs from the B6.TCR-β^–/–/^–/– mice, we used a purification kit for CD8⁺ DC that depletes CD90- (Thy-1.2), CD45R- (B220), and CD49b-positive cells, followed by positive magnetic bead selection of CD8⁺ cells. This cell fraction contained at a minimum 60% CD8⁺ DCs (MHC class II⁺, CD11c⁺, and CD8⁺). These enriched cells, when injected i.v. (10⁵ CD8⁺ DC/inoculum), were sufficient to support the development of AHR-suppressive V4⁺ T cells in B6.CD8^–/– mice (Fig. 5c). In contrast, neither CD8^– splenocytes nor enriched CD8^– DCs (Fig. 5c) nor splenic CD8⁺ β T cells (from B6.TCR-^–/– donors) had any reconstituting activity (Fig. 5d), albeit provided in larger numbers. These data suggest that CD8⁺ DCs function as accessory cells in the development of the V4⁺ AHR suppressor T cells, whereas other DC types or T cells do not.

View larger version (46K): [in this window] [in a new window]   FIGURE 5. AHR in T cell-deficient recipients reconstituted with genetically CD8-deficient V4+ cells from "CD8-restored donors" after i.p. sensitization and airway challenge with OVA (2ip3N protocol). Lung resistance (RL) and dynamic compliance (Cdyn) as percentage changes from saline controls in relation to increasing doses of aerosolized MCh are shown. Recipients are B6.TCR-V4/6–/– mice and donors are B6.CD8–/– mice. Treatments are as follows: none (NT); systemic sensitization and airway challenge (2ip3N); and 2ip3N plus V4+ cells (2ip3N + V4 cells) from 2ip3N-treated donors. The donors of the V4+ cells either remained nonrestored or were restored with splenocytes from 2ip-treated B6.TCR-β–/–/–/– mice, including the following fractions: whole spleen (a); magnetic bead-selected CD8+ or CD8– splenocytes (b); or DC-enriched, magnetic bead-selected CD8+ or CD8– DCs (c). Donors of V4+ cells were restored with nylon wool-nonadherent, magnetic bead-selected CD8+ T cells from 2ip-treated B6.TCR-–/– mice (d). a, Recipients were untreated or treated with 2ip3N plus V4+ cells from nonrestored donors or donors that were restored with whole spleen (*, 2ip3N plus V4 cells compared with 2ip3N plus V4 cells from a donor restored with whole spleen; #, nontreated compared with 2ip3N plus V4 cells from a nonrestored donor). b, Untreated or treated with 2ip3N plus V4+ cells from 2ip3N-treated donors restored with CD8-enriched or CD8-depleted splenocytes (*, nontreated compared with 2ip3N plus V4 cells from donors reconstituted with CD8– splenocytes; #, 2ip3N plus V4 cells from donors reconstituted with CD8– splenocytes compared with 2ip3N plus V4 cells from donor reconstituted with CD8+ splenocytes). c, Untreated or treated with 2ip3N plus V4+ cells from donors restored with CD8+ or CD8– splenic DCs or with non-DCs (mostly CD8–) (*, nontreated compared with 2ip3N plus V4 cells from donor restored with non-DCs; #, treated with CD8– DCs; $, treated with 2ip3N plus V4 cells from donor restored with CD8+ DCs compared with 2ip3N plus V4 cells from donor restored with non-DCs; #, treated with CD8– DCs). d, Untreated or treated with 2ip3N or 2ip3N plus V4+ cells from donors restored with CD8+ T cells (*, nontreated compared with 2ip3N treatment; #, nontreated compared with 2ip3N plus V4 cells from donors reconstituted with CD8+ T cells; +, no significant difference in RL between 2ip3N and 2ip3N plus V4 cells from donors reconstituted with CD8+ T cells, with the exception of the response to 12.5 mg of MCh). One symbol (*, #, +, or $) = p < 0.05; two symbols = p < 0.01; three symbols = p < 0.001.

AHR-enhancing T cells also depend on CD8, and enriched CD8⁺ DCs enable their development

V1⁺ T cells enhance AHR as shown by cell depletion and transfer experiments (10). In fact, the development of AHR in young mice depends on these cells (10, 11), whereas AHR in older mice can develop in their absence while remaining subject to the influence of AHR-suppressive T cells (5, 14). In contrast to the AHR suppressors, the AHR-enhancing T cells develop spontaneously without the need for sensitization or challenge of the cell donors (11). However, we found that B6.CD8^–/– mice were also unable to produce these AHR enhancers (Fig. 6a). Therefore, we tested whether reconstituting these mice with CD8⁺ non-T cells would also restore development of the AHR-enhancing T cells in the absence of any sensitization or challenge. Indeed, we found that reconstituting B6.CD8^–/– mice with the CD8⁺ splenocyte fraction of B6.TCR-β^–/–/^–/– mice (Fig. 6b) or with small numbers of enriched CD8⁺ DC (Fig. 6c), restored their ability to produce the AHR enhancers, whereas the CD8^– splenocyte fraction had no effect (Fig. 6b). Because restoration of the AHR enhancers occurred without sensitizing the source of the DC and without sensitizing or challenging the donors of the T cells, this result suggested that interactions between CD8⁺ DC and T cells are an integral part of normal and spontaneous T cell development.

View larger version (28K): [in this window] [in a new window]   FIGURE 6. AHR in T cell-deficient recipients reconstituted with V1+ cells from normal or CD8-deficient mice or with CD8-deficient V1+ cells from "CD8-restored donors" after systemic sensitization and airway challenge with OVA (2ip3N protocol). Lung resistance (RL) and dynamic compliance (Cdyn) as percentage changes from saline controls in relation to increasing doses of aerosolized MCh as shown. Recipients were B6.TCR-–/– mice and donors were B6 or B6.CD8–/– mice. Treatment of recipients was as follows: none (NT); i.p. sensitization and airway challenge (2ip3N), 2ip3N plus selected purified V1+ cells from nontreated donors, 2ip3N treatment plus V1+ cells from nontreated B6.CD8–/– mice restored with magnetic bead-selected CD8+; CD8– splenocytes from 2ip-treated B6.TCR-β–/–/–/– mice; or 2ip3N plus V1+ cells from nontreated B6.CD8–/– mice restored with DC-enriched, magnetic bead-selected CD8+ or CD8– DCs from 2ip-treated B6.TCR-β–/–/–/– mice. a, Recipients after 2ip3N treatment or 2ip3N treatment plus receipt of V1+ cells from B6 or B6.CD8–/– donors (*, 2ip3N compared with 2ip3N plus V1+ cells from B6 donors; #, 2ip3N plus V1+ cells from B6 donors compared with 2ip3N plus V1+ cells from B6.CD8–/– donors). b, Recipients after 2ip3N treatment or 2ip3N treatment plus receipt of V1+ cells from B6.CD8–/– donors restored with CD8+ or CD8– spleen cells from nontreated B6.TCR-β–/–/–/– mice (*, 2ip3N compared with 2ip3N plus V1+ cells from B6.CD8–/– donors restored with CD8+ spleen cells; #, 2ip3N plus V1+ cells from B6.CD8–/– donors restored with CD8+ spleen cells compared with 2ip3N plus V1+ cells from B6.CD8–/– donors restored with CD8– spleen cells). c, Recipients after 2ip3N treatment or 2ip3N treatment plus receipt of V1+ cells from nontreated B6.CD8–/– donors restored with CD8+ or CD8– splenic DCs from nontreated B6.TCR-β–/–/–/– mice (*, 2ip3N compared with 2ip3N plus V1+ cells from B6.CD8–/– donors restored with CD8+ DCs; #, 2ip3N plus V1+ cells from B6.CD8–/– donors restored with CD8+ DCs compared with 2ip3N plus V1+ cells from B6.CD8–/– donors restored with CD8– DCs). One symbol (*, #, or +) = p < 0.05; two symbols = p < 0.01; three symbols = p < 0.001.

Are the encounters between T cells and CD8⁺ DC in the spleen functionally relevant?

In B6.CD8^–/– mice T cells are still present in the PALS (Fig. 3f), whereas in T cell-deficient mice (B6.TCR-β^–/–/^–/–) CD8⁺ non-T cells still populate the general vicinity of the central arterioles (Fig. 3g). Although there likely are quantitative differences in cell distributions by comparison with wild-type mice, this indicates that neither cell type absolutely requires the other to accumulate at this site.

To test whether the transferred CD8⁺ DCs that restore the development of AHR-regulatory T cells in B6.CD8^–/– mice indeed appear in the spleen and engage T cells, we transferred purified CD8⁺ non-T cells from a B6.TCR-β^–/–/^–/– spleen in larger numbers (1 x 10⁶) to facilitate their detection in situ. Five days later, when the regulatory T cells are normally harvested, the CD8⁺ non-T cells were indeed detectable in the PALS of the B6.CD8^–/– mice proximal to and frequently in direct contact with the endogenous T cells (Fig. 3k). Thus, the transferred CD8⁺ non-T cells (in the B6.CD8^–/– mice) accumulated and encountered T cells in the same splenic location as the endogenous CD8⁺ non-T cells in the B6.TCR-β^–/– mice. Because these transferred cells also enable splenic T cell function (Figs. 5 and 6), their interactions with T cells in the spleen are likely to be critical for the functional development of the AHR regulators.

Finally, because development of the AHR-suppressive T cells in the spleen depends on donor sensitization with OVA, we also examined the histological distribution of i.p. injected OVA/alum (Fig. 3l). We found OVA/alum in the spleen in aggregates or contained within CD8^– phagocytes, but not within CD8⁺ non-T cells or T cells. However, both the CD8⁺ non-T cells and the T cells were seen in proximity/contact with the OVA/alum in the spleen (Fig. 3l).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Because innate T cells can become powerful modulators of AHR, it is important to understand their development. We have examined the development of two T cell types in mice that are capable of either suppressing or enhancing allergen-induced AHR. These cells express V4 and V1, respectively, and they are present in the lung and in secondary lymphoid tissues (10, 16). The AHR-suppressive V4⁺ cells are induced by allergen sensitization and challenge (15) whereas the AHR-enhancing V1⁺ cells develop spontaneously (11). In this study we report three observations related to the development of these two cell-types, namely that the process requires CD8, that the T cells encounter CD8⁺ non-T cells that appear to be DCs in the splenic PALS, and that enriched CD8⁺ DCs from the spleen can restore the development of these two T cell types when transferred into genetically CD8-deficient mice.

Crosstalk between T cells and DCs occurs in vitro (20, 21, 22, 24), but little is known about their cellular interactions in the spleen and other lymphoid tissues. In mice lacking β T cells (B6.TCR-β^–/–), we found many of the T cells within the splenic PALS in proximity to or in contact with what appear to be CD8⁺ DCs based on morphology, phenotype, and tissue location. In wt mice (B6) there are fewer T cells. These still accumulate in the PALS (Fig. 3a), but due to their smaller numbers and perhaps also because of competition with the β T cells (37, 38), one might expect to find overall fewer contacts with DCs. However, in mice lacking CD8 that contain β T cells, the T cells were found in the proximity of what appeared to be endogenous DEC-205⁺ DCs in the PALS, and they were not prevented from contact with transferred splenic CD8⁺ non-T cells, which are probably CD8⁺ DCs (Fig. 3k). Contact with endogenous splenic CD8⁺ non-T cells seem to occur continuously, as there was little change in frequency following OVA sensitization/challenge, and both V1⁺ cells and V4⁺ cells took part. Likewise, enriched CD8⁺ DCs can restore the development of both V1⁺ AHR enhancers and V4⁺ AHR suppressors. Our preliminary data (not shown) suggest that the transfer of CD8⁺ DCs drives the T cells into cell cycle (C.L. Roark and W. K. Born, unpublished observations). However, the CD8⁺ DCs appear to exert their effect at an early stage in the development of the AHR-modulating T cells, and these interactions alone seem insufficient to explain the functional differentiation of the AHR enhancers and suppressors. Notably, development of the suppressors requires airway challenge and thus may depend on a lung-derived signal (11, 15).

Our data suggest a strict requirement for CD8 in the development of the AHR-regulatory T cells, although these cells themselves need not express CD8. A role for CD8 "in trans" in T cell development has not been reported (39). Because transferred enriched CD8⁺ DC can restore development of the AHR modulators in the absence of other CD8⁺ cells in the recipients, it seems likely that the DC-expressed CD8 satisfies the requirement for CD8 although other more complex scenarios cannot be excluded. A direct mechanism would be interesting because, although CD8⁺ DCs in the lymphoid tissues have been characterized as a phenotypically and functionally distinct population (31), no functional significance has been assigned to the CD8 they express. In fact, in one study comparing the largely overlapping populations of CD8⁺ and DEC-205⁺ DCs for their ability to support β T cell responses, DC-expressed CD8 appeared to be of no consequence because CD8⁺ DCs could be replaced with DEC-205⁺ DCs without change (40). Our findings contrast with this study inasmuch as endogenous DEC-205⁺ cells, which are plentiful in the spleens of CD8^– mice and colocalize here with T cells (Fig. 3j), failed to support the development of the AHR-modulating T cells. Thus, DC-expressed CD8 might have a function after all in connection with the development of T cells, perhaps as an activating receptor (41, 42).

CD8⁺ DCs, which reside in the lymphoid tissues (28, 29), have been found to receive Ags and activating signals from phagocytes that shuttle from peripheral tissues such as skin and lung to the secondary lymphoid organs (43, 44), to excel at Ag-cross-presentation, and to prime naive β T cells to become Ag-specific CTLs and Th1 cells (45, 46, 47). To our knowledge, they have not previously been connected with T cells. However, a dependence on CD8⁺ DCs aligns the T cells with the above-mentioned types of β T cells (CD8⁺ and Th1) and sets them apart from others (Th2) and from NK cells, neither of which depend on CD8⁺ DCs although they require DC priming (25, 48). We have not yet determined which factors might be involved in the DC- T interaction. Candidates include TNF- and IL-15, both of which are produced by CD8⁺ DCs, and TNF-, because the function and development of AHR-regulatory T cells depends on it (49, 50), and IL-15, because it supports homeostatic expansion of T cells (37). Incidentally, IL-15 is "trans"-presented by DCs, requires DC-lymphocyte contact, and also is essential in the "priming" of NK cells (48).

Connecting the observations reported in this study, we propose that developing T cells must interact with CD8⁺ DCs in the lymphoid tissues as an early step on their way to functional competence (e.g., toward becoming AHR modulators). Because this interaction appears to involve direct cell contact, because CD8 is absolutely required for T cell development, and because development can take place when the only available CD8 appears to be expressed by the DCs, we envisage that the DC-expressed CD8 molecules might engage ligands on the T cells they contact. Known ligands for CD8 are MHC class I or class I-like molecules (12, 51). We have not yet examined AHR enhancement but we found that AHR suppression by V4⁺ T cells indeed depends on MHC class I expression (L. Cook and W. K. Born, unpublished observations). Thus, our data are consistent with the scenario outlined but fall short of proving it. However, in another study with naturally occurring regulatory T cells whose functional activation requires both CD8 and MHC I, a similar cell-cell interaction was proposed (52).

The interaction between T cells and CD8⁺ DCs documented here likely represents only one of several steps in the development of the T cells. As both AHR-suppressive and AHR-enhancing types depend on CD8⁺ DCs, their functional differences might be determined elsewhere, earlier or later in their development (53). Additional accessory cells such as the CD8^– phagocytes shown in Fig. 3l, which engulf OVA/alum particles and appear following sensitization in the splenic PALS near the CD8⁺ DCs and the T cells, are perhaps involved as well (43, 44). These cells or cells from the challenged lung might mediate the functional differentiation of the splenic AHR suppressors, which require induction by OVA sensitization and challenge (15).

	Acknowledgments

 
We thank Drs. Katsuyuki Takeda, M. Kemal Aydintug, and Jena French for advice and support, Drs. Philippa Marrack and Michael Lahn for discussion, and Joshua Loomis and Bill Townend for assistance with cell sorts and fluorescence microscopy.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants AI40611 and HL65410 to W.K.B., AI44920 and AI063400 to R.L.O., and HL36577 to E.W.G.

² Address correspondence and reprint requests to Dr. W. K. Born, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: bornw{at}njc.org

³ Abbreviations used in this paper: AHR, airway hyperresponsiveness; DC, dendritic cell; MCh, methacholine; PALS, periarteriolar lymphoid sheath.

Received for publication December 11, 2007. Accepted for publication April 24, 2008.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Wills-Karp, M.. 1999. Immunologic basis of antigen-induced airway hyperresponsiveness. Annu. Rev. Immunol. 17: 255-281. [Medline]
2. Grunig, G., M. Warnock, A. E. Wakil, R. Venkayya, F. Brombacher, D. M. Rennick, D. Sheppard, M. Mohrs, D. D. Donaldson, R. M. Locksley, D. B. Corry. 1998. Requirement for IL-13 independently of IL-4 in experimental asthma. Science 282: 2261-2263. [Abstract/Free Full Text]
3. McMenamin, C., C. Pimm, M. McKersey, P. G. Holt. 1994. Regulation of IgE responses to inhaled antigen in mice by antigen-specific T cells. Science 265: 1869-1871. [Abstract/Free Full Text]
4. Zuany-Amorim, C., C. Ruffie, S. Haile, B. B. Vargaftig, P. Pereira, M. Pretolani. 1998. Requirement for T cells in allergic airway inflammation. Science 280: 1265-1267. [Abstract/Free Full Text]
5. Lahn, M., A. Kanehiro, K. Takeda, A. Joetham, J. Schwarze, G. Koehler, R. O'Brien, E. W. Gelfand, W. Born. 1999. Negative regulation of airway responsiveness that is dependent on T cells and independent of β T cells. Nat. Med. 5: 1150-1156. [Medline]
6. Akbari, O., P. Stock, E. Meyer, M. Kronenberg, S. Sidobre, T. Nakayama, M. Taniguchi, M. J. Grusby, R. H. DeKruyff, D. T. Umetsu. 2003. Essential role of NKT cells producing IL-4 and IL-13 in the development of allergen-induced airway hyperreactivity. Nat. Med. 9: 582-588. [Medline]
7. Hadeiba, H., R. M. Locksley. 2003. Lung CD25 CD4 regulatory T cells suppress type 2 immune responses but not bronchial hyperreactivity. J. Immunol. 170: 5502-5510. [Abstract/Free Full Text]
8. Lahn, M., A. Kanehiro, Y.-S. Hahn, J. M. Wands, M. K. Aydintug, R. L. O'Brien, E. W. Gelfand, W. K. Born. 2004. Aerosolized anti-T-cell-receptor antibodies are effective against airway inflammation and hyperreactivity. Int. Arch. Allergy Immunol. 134: 49-55. [Medline]
9. Hamelmann, E., A. Oshiba, J. Paluh, K. Bradley, J. Loader, T. A. Potter, G. L. Larsen, E. W. Gelfand. 1996. Requirement for CD8⁺ T cells in the development of airway hyperresponsiveness in a murine model of airway sensitization. J. Exp. Med. 183: 1719-1729. [Abstract/Free Full Text]
10. Hahn, Y.-S., C. Taube, N. Jin, L. Sharp, J. M. Wands, M. K. Aydintug, M. Lahn, S. A. Huber, R. L. O'Brien, E. W. Gelfand, W. K. Born. 2004. Different potentials of T cell subsets in regulating airway responsiveness: V1⁺ cells, but not V4⁺ cells, promote airway hyperreactivity, TH2 cytokines, and airway inflammation. J. Immunol. 172: 2894-2902. [Abstract/Free Full Text]
11. Jin, N., N. Miyahara, C. L. Roark, J. D. French, M. K. Aydintug, J. L. Matsuda, L. Gapin, R. L. O'Brien, E. W. Gelfand, W. K. Born. 2007. Airway hyperresponsiveness through synergy of T cells and NKT cells. J. Immunol. 179: 2961-2968. [Abstract/Free Full Text]
12. Lahn, M., A. Kanehiro, K. Takeda, J. Terry, Y.-S. Hahn, M. K. Aydintug, A. Konowal, K. Ikuta, R. L. O'Brien, E. W. Gelfand, W. K. Born. 2002. MHC class I-dependent V4⁺ pulmonary T cells regulate β T cell-independent airway responsiveness. Proc. Natl. Acad. Sci. USA 99: 8850-8855. [Abstract/Free Full Text]
13. Hahn, Y.-S., C. Taube, N. Jin, K. Takeda, J.-W. Park, J. M. Wands, M. K. Aydintug, C. L. Roark, M. Lahn, R. L. O'Brien, et al 2003. V4⁺ T cells regulate airway hyperreactivity to methacholine in ovalbumin-sensitized and challenged mice. J. Immunol. 171: 3170-3178. [Abstract/Free Full Text]
14. Cui, Z.-H., A. Joetham, M. K. Aydintug, W. K. Born, E. W. Gelfand. 2003. Reversal of established allergic airway hyperreactivity by long-term allergen challenge depends on / T cells. Am. J. Respir. Crit. Care Med. 168: 1324-1332. [Abstract/Free Full Text]
15. Jin, N., C. Taube, L. Sharp, Y.-S. Hahn, X. Yin, J. M. Wands, C. L. Roark, R. L. O'Brien, E. W. Gelfand, W. K. Born. 2005. Mismatched antigen prepares T cells for suppression of airway hyperresponsiveness. J. Immunol. 174: 2671-2679. [Abstract/Free Full Text]
16. Wands, J. M., C. L. Roark, M. K. Aydintug, N. Jin, Y.-S. Hahn, L. Cook, X. Yin, J. Dalporto, M. Lahn, D. M. Hyde, et al 2005. Distribution and leukocyte contacts of T cells in the lung. J. Leukocyte Biol. 78: 1086-1096. [Abstract/Free Full Text]
17. Banchereau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392: 245-252. [Medline]
18. Ismaili, J., V. Olislagers, M. Poupot, J. J. Fournie, M. Goldman. 2002. Human T cells induce dendritic cell maturation. Clin. Immunol. 103: 296-302. [Medline]
19. Leslie, D. S., M. S. Vincent, F. M. Spada, H. Das, M. Sugita, C. T. Morita, M. B. Brenner. 2002. CD1-mediated / T cell maturation of dendritic cells. J. Exp. Med. 196: 1575-1584. [Abstract/Free Full Text]
20. Collins, C., J. Wolfe, K. Roessner, C. Shi, L. H. Sigal, R. C. Budd. 2005. Lyme arthritis synovial T cells instruct dendritic cells via Fas ligand. J. Immunol. 175: 5656-5665. [Abstract/Free Full Text]
21. Dieli, F., N. Caccamo, S. Meraviglia, J. Ivanyi, G. Sireci, C. T. Bonanno, V. Ferlazzo, C. La Mendola, A. Salerno. 2004. Reciprocal stimulation of T cells and dendritic cells during the anti-mycobacterial immune response. Eur. J. Immunol. 34: 3227-3235. [Medline]
22. Devilder, M.-C., S. Maillet, I. Bouyge-Moreau, E. Donnadieu, M. Bonneville, E. Scotet. 2006. Potentiation of antigen-stimulated V9V2 T cell cytokine production by immature dendritic cells (DC) and reciprocal effect on DC maturation. J. Immunol. 176: 1386-1393. [Abstract/Free Full Text]
23. Kunzmann, V., E. Kretzschmar, T. Herrmann, M. Wilhelm. 2004. Polyinosinic-polycytidylic acid-mediated stimulation of human T cells via CD11c dendritic cell-derived type I interferons. Immunology 112: 364-368. [Medline]
24. Conti, L., R. Casetti, M. Cardone, B. Varano, A. Martino, F. Belardelli, F. Poccia, S. Gessani. 2005. Reciprocal activating interaction between dendritic cells and pamidronate-stimulated T cells: role of CD86 and inflammatory cytokines. J. Immunol. 174: 252-260. [Abstract/Free Full Text]
25. Maldonado-Lopez, R., T. De Smedt, P. Michel, J. Godfroid, B. Pajak, C. Heirman, K. Thielemans, O. Leo, J. Urbain, M. Moser. 1999. CD8⁺ and CD8^– subclasses of dendritic cells direct the development of distinct T helper cells in vivo. J. Exp. Med. 189: 587-592. [Abstract/Free Full Text]
26. Allan, R. S., C. M. Smith, G. T. Belz, A. L. van Lint, L. M. Wakim, W. R. Heath, F. R. Carbone. 2003. Epidermal viral immunity induced by CD8⁺ dendritic cells but not by Langerhans cells. Science 301: 1925-1928. [Abstract/Free Full Text]
27. Wilson, N. S., G. M. N. Behrens, R. J. Lundie, C. M. Smith, J. Waithman, L. Young, S. P. Forehan, A. Mount, R. J. Steptoe, K. D. Shortman, et al 2006. Systemic activation of dendritic cells by Toll-like receptor ligands or malaria infection impairs cross-presentation and antiviral immunity. Nat. Immunol. 7: 165-172. [Medline]
28. Dakic, A., Q.-X. Shao, A. D'Amico, M. O'Keefe, W.-F. Chen, K. Shortman, L. Wu. 2004. Development of the dendritic cell system during mouse ontogeny. J. Immunol. 172: 1018-1027. [Abstract/Free Full Text]
29. Heath, W. R., G. T. Belz, G. M. N. Behrens, C. M. Smith, S. P. Forehan, I. A. Parish, G. M. Davey, N. S. Wilson, F. R. Carbone, J. A. Villadangos. 2004. Cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens. Immunol. Rev. 199: 9-26. [Medline]
30. Pillarisetty, V. G., S. C. Katz, J. I. Bleier, A. B. Shah, R. P. Dematteo. 2005. Natural killer dendritic cells have both antigen presenting and lytic function and in response to CpG produce IFN- via autocrine IL-12. J. Immunol. 174: 2612-2618. [Abstract/Free Full Text]
31. Shortman, K., S. H. Naik. 2007. Steady-state and inflammatory dendritic cell development. Nat. Rev. Immunol. 7: 19-30. [Medline]
32. Sunaga, S., K. Maki, Y. Komagata, J.-I. Miyazaki, K. Ikuta. 1997. Developmentally ordered V-J recombination in mouse T cell receptor locus is not perturbed by targeted deletion of the V4 gene. J. Immunol. 158: 4223-4228. [Abstract]
33. Dent, A. L., L. A. Matis, F. Hooshmand, S. M. Widacki, J. A. Bluestone, S. M. Hedrick. 1990. Self-reactive T cells are eliminated in the thymus. Nature 343: 714-719. [Medline]
34. Heilig, J. S., S. Tonegawa. 1987. T-cell gene is allelically but not isotypically excluded and is not required in known functional T-cell subsets. Proc. Natl. Acad. Sci. USA 84: 8070-8074. [Abstract/Free Full Text]
35. Pereira, P., D. Gerber, S. Y. Huang, S. Tonegawa. 1995. Ontogenic development and tissue distribution of V1-expressing / T lymphocytes in normal mice. J. Exp. Med. 182: 1921-1930. [Abstract/Free Full Text]
36. Miyahara, N., B. J. Swanson, K. Takeda, C. Taube, S. Miyahara, T. Kodama, A. Dakhama, V. L. Ott, E. W. Gelfand. 2004. Effector CD8⁺ T cells mediate inflammation and airway hyper-responsiveness. Nat. Med. 10: 865-869. [Medline]
37. French, J. D., C. L. Roark, W. K. Born, R. L. O'Brien. 2005. T cell homeostasis is established in competition with β T cells and NK cells. Proc. Natl. Acad. Sci. USA 102: 14741-14746. [Abstract/Free Full Text]
38. Willis, R. A., J. W. Kappler, P. C. Marrack. 2006. CD8 T cell competition for dendritic cells in vivo is an early event in activation. Proc. Natl. Acad. Sci. USA 103: 12063-12068. [Abstract/Free Full Text]
39. Rammensee, H.-G., M. J. Bevan, P. J. Fink. 1985. Antigen specific suppression of T-cell responses: the veto concept. Immunol. Today 6: 41-43.
40. Kronin, V., D. Vremec, K. Winkel, B. J. Classon, R. G. Miller, T. W. Mak, K. Shortman, G. Suss. 1997. Are CD8⁺ dendritic cells (DC) veto cells? The role of CD8 on DC in DC development and in the regulation of CD4 and CD8 T cell responses. Int. Immunol. 9: 1061-1064. [Abstract/Free Full Text]
41. Attinger, A., L. Devine, Y. Wang-Zhu, D. Martin, J.-h. Wang, E. L. Reinherz, M. Kronenberg, H. Cheroutre, P. Kavathas. 2005. Molecular basis for the high affinity interaction between the thymic leukemia antigen and the CD8 molecule. J. Immunol. 174: 3501-3507. [Abstract/Free Full Text]
42. Kern, P., R. E. Hussey, R. Spoerl, E. L. Reinherz, H.-C. Chang. 1999. Expression, purification and functional analysis of murine ectodomain fragments of CD8 and CD8β dimers. J. Biol. Chem. 274: 27237-27243. [Abstract/Free Full Text]
43. Belz, G. T., C. M. Smith, L. Kleinert, P. Reading, A. Brooks, K. Shortman, F. R. Carbone, W. R. Heath. 2004. Distinct migrating and nonmigrating dendritic cell populations are involved in MHC class I-restricted antigen presentation after lung infection with virus. Proc. Natl. Acad. Sci. USA 101: 8670-8675. [Abstract/Free Full Text]
44. Allan, R. S., J. Waithman, S. Bedoui, C. M. Jones, J. A. Villadangos, Y. Zhan, A. M. Lew, K. Shortman, W. R. Heath, F. R. Carbone. 2006. Migratory dendritic cells transfer antigen to a lymph node-resident dendritic cell population for efficient CTL priming. Immunity 25: 153-162. [Medline]
45. den Haan, J. M. M., S. M. Lehar, M. J. Bevan. 2000. CD8⁺ but not CD8^– dendritic cells cross-prime cytotoxic T cells in vivo. J. Exp. Med. 192: 1685-1695. [Abstract/Free Full Text]
46. Belz, G. T., K. Shortman, M. J. Bevan, W. R. Heath. 2005. CD8⁺ dendritic cells selectively present MHC class I-restricted noncytolytic viral and intracellular bacterial antigens in vivo. J. Immunol. 175: 196-200. [Abstract/Free Full Text]
47. Belz, G. T., C. M. Smith, D. Eichner, K. Shortman, G. Karupiah, F. R. Carbone, W. R. Heath. 2004. Cutting edge: conventional CD8⁺ dendritic cells are generally involved in priming CTL immunity to viruses. J. Immunol. 172: 1996-2000. [Abstract/Free Full Text]
48. Lucas, M., W. Schachterle, K. Oberle, P. Aichele, A. Diefenbach. 2007. Dendritic cells prime natural killer cells by trans-presenting interleukin 15. Immunity 26: 503-517. [Medline]
49. Kanehiro, A., M. Lahn, M. J. Makela, A. Dakhama, M. Fujita, A. Joetham, R. J. Mason, W. Born, E. W. Gelfand. 2001. Tumor necrosis factor- negatively regulates airway hyperresponsiveness through T cells. Am. J. Respir. Crit. Care Med. 164: 2229-2238. [Abstract/Free Full Text]
50. Kanehiro, A., M. Lahn, M. J. Makela, A. Dakhama, A. Joetham, Y.-H. Rha, W. Born, E. W. Gelfand. 2002. Requirement for the p75 TNF- receptor 2 in the regulation of airway hyperresponsiveness by T cells. J. Immunol. 169: 4190-4197. [Abstract/Free Full Text]
51. Leishman, A. J., O. V. Naidenko, A. Attinger, F. Koning, C. J. Lena, Y. Xiong, H.-C. Chang, E. Reinherz, M. Kronenberg, H. Cheroutre. 2001. T cell responses modulated through interaction between CD8 and the nonclassical MHC class I molecule, TL. Science 294: 1936-1939. [Abstract/Free Full Text]
52. Joetham, A., K. Takeda, N. Miyahara, S. Matsubara, H. Ohnishi, T. Koya, A. Dakhama, E. W. Gelfand. 2007. Activation of naturally occurring lung CD4⁺CD25⁺ regulatory T cells requires CD8 and MHC I interaction. Proc. Natl. Acad. Sci. USA 104: 15057-15062. [Abstract/Free Full Text]
53. O'Brien, R. L., C. L. Roark, N. Jin, M. K. Aydintug, J. D. French, J. L. Chain, J. M. Wands, M. Johnston, W. K. Born. 2007. T cell receptors: functional correlations. Immunol. Rev. 215: 77-88. [Medline]

This article has been cited by other articles:

				Y. Huang, N. Jin, C. L. Roark, M. K. Aydintug, J. M. Wands, H. Huang, R. L. O'Brien, and W. K. Born The Influence of IgE-Enhancing and IgE-Suppressive {gamma}{delta} T Cells Changes with Exposure to Inhaled Ovalbumin J. Immunol., July 15, 2009; 183(2): 849 - 855. [Abstract] [Full Text] [PDF]

				N. Jin, C. L. Roark, N. Miyahara, C. Taube, M. K. Aydintug, J. M. Wands, Y. Huang, Y.-S. Hahn, E. W. Gelfand, R. L. O'Brien, et al. Allergic Airway Hyperresponsiveness-Enhancing {gamma}{delta} T Cells Develop in Normal Untreated Mice and Fail to Produce IL-4/13, Unlike Th2 and NKT Cells J. Immunol., February 15, 2009; 182(4): 2002 - 2010. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) 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 Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cook, L. Articles by Born, W. K. Search for Related Content PubMed PubMed Citation Articles by Cook, L. Articles by Born, W. K.