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 Trobonjaca, Z. Articles by Reimann, J. Search for Related Content PubMed PubMed Citation Articles by Trobonjaca, Z. Articles by Reimann, J.

MHC-II-Independent CD4⁺ T Cells Induce Colitis in Immunodeficient RAG^-/- Hosts¹

Zlatko Trobonjaca^*, Frank Leithäuser, Peter Möller, Horst Bluethmann, Yasuhiko Koezuka, H. Robson MacDonald^¶ and Jörg Reimann^2,*

Departments of ^* Medical Microbiology and Immunology and Pathology, University of Ulm, Ulm, Germany; Roche Genetics, F. Hoffmann LaRoche, Basel, Switzerland; Kirin Brewery, Pharmaceutical Research Laboratory, Gunma, Japan; and ^¶ Ludwig Institute for Cancer Research, Epalinges, Switzerland

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD4⁺ T cells from either normal C57BL/6 (B6) or MHC-II-deficient (A^-/- or A^-/-) B6 donor mice engrafted into congenic immunodeficient RAG1^-/- B6 hosts induced an aggressive inflammatory bowel disease (IBD). Furthermore, CD4⁺ T cells from CD1d^-/- knockout (KO) B6 donor mice but not those from MHC-I^-/- (homozygous transgenic mice deficient for ₂-microglobulin) KO B6 mice induced a colitis in RAG^-/- hosts. Abundant numbers of in vivo activated (CD69^highCD44^highCD28^high) NK1⁺ and NK1^- CD4⁺ T cells were isolated from the inflamed colonic lamina propria (cLP) of transplanted mice with IBD that produced large amounts of TNF- and IFN- but low amounts of IL-4 and IL-10. IBD-associated cLP Th1 CD4⁺ T cell populations were polyclonal and MHC-II-restricted when derived from normal B6 donor mice, but oligoclonal and apparently MHC-I-restricted when derived from MHC-II-deficient (A^-/- or A^-/-) B6 donor mice. cLP CD4⁺ T cell populations from homozygous transgenic mice deficient for ₂-microglobulin KO B6 donor mice engrafted into RAG^-/- hosts were Th2 and MHC-II restricted. These data indicate that MHC-II-dependent as well as MHC-II-independent CD4⁺ T cells can induce a severe and lethal IBD in congenic, immunodeficient hosts, but that the former need the latter to express its IBD-inducing potential.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The adoptive transfer of CD4⁺ CD3⁺ T cells into congenic, immunodeficient hosts induces an inflammatory bowel disease (IBD),³ the major manifestation of which is a severe pancolitis. This has been demonstrated in H-2^d mice of the BALB/c background (1, 2, 3, 4, 5) and in H-2^b mice of the C57BL/6 (B6) background (6, 7, 8). The key factors for the development of an IBD in these models are an inducing CD3⁺CD4⁺ T cell subset, a congenic, severely immunodeficient host, and an intact gut flora of the host. A defect in the immunoregulation of mucosal T cell responses is supposed to play a key role in the pathogenesis of colitis in these models, with exaggerated IFN- and TNF- responses as major mediators of this disease (reviewed in (9, 10). These pathogenic T cell responses may be driven by Ags of the intestinal flora to which these T cells are normally tolerant. In the host developing colitis, neither the inducing CD4⁺ T cell subset nor the stimulus that drives the Th1-biased T cell activation in the mucosa has been identified. Splenic CD4⁺ T cells with a CD45RB^high surface phenotype have an enhanced IBD-inducing potential in H-2^d C.B-17^scid/scid (SCID) mice (reviewed in Ref. 11). It is unresolved which IBD-inducing CD4⁺ T cell subset(s) the CD45RB^high surface phenotype identifies, and it has been shown that CD45RB^high as well as CD45RB^lowCD4⁺ T cell can induce a colitis in this adoptive transfer system (8, 12). IBD-associated effector T cell populations accumulating in the inflamed colonic lamina propria (cLP) in different mouse models can be conventional CD4^{+ +} T cells, ⁺ and ⁺ T cells (13), CD8^{+ +} T cells (14), or unusual TCR^-+ T cells (15). Key questions of the pathogenesis of T cell-induced IBD are unresolved. Preclinical mouse models offer an attractive approach to clarify some aspects of these common and debilitating diseases that may pave the way for the rational design of novel therapeutic approaches.

Development of epitope recognition of most CD4⁺ T cells is MHC-II-dependent. A small subset of MHC-II-independent CD4⁺ T cells is present in normal and MHC-II-deficient (A^-/- or A^-/- knockout (KO)) mice. MHC-II-independent CD4⁺ T cells in A^-/- mice are thymically derived, appear early in ontogeny, localize preferentially to the B rather than to the T cell areas in peripheral lymphoid organs, exhibit the phenotype of resting or activated memory T cells, and have a diverse TCR repertoire that is potentially functional (16, 17). These T cell populations are heterogeneous with respect to phenotype, array of peptides and/or glycolipids recognized, and restriction elements used for specific recognition (18, 19, 20, 21, 22). Only one subset within the MHC-II-independent CD4⁺ T cells, i.e., CD1d-restricted NK1⁺ T cells expressing the NK1 marker (NKT) cells is well defined (reviewed in Refs. 23, 24). NKT cells express the semi-invariant V14J281 V8.2 TCR, bear the NK1.1 marker (in appropriate mouse strains), are found mainly in thymus and liver, rapidly produce the cytokines IFN- and IL-4 after stimulation, and recognize the glycolipid -galactosyl ceramide (-GalCer) in the context of CD1d (17, 25, 26, 27, 28, 29, 30, 31). MHC-II-independent, CD1d-restricted NKT cells are absent from CD1d^-/- KO mice (32, 33, 34). The presence and function of MHC-II-independent CD4⁺ T cells in the gut is not clear. Although NK-like T cells have been found in the intraepithelial compartment of the small intestine of mice (35), NK1⁺ NKT cells are absent from the intestinal intraepithelial lymphocyte and lamina propria lymphocyte (LPL) populations (36). Recently, CD1d-glycolipid tetramer staining has revealed NK1^-CD4^-CD8^- tetramer-positive T cells with the characteristic semi-invariant TCR in lymph nodes and the small intestine (22, 37, 38).

We have transferred splenic CD4⁺ T cells developing in a normal (MHC-I, MHC-II, CD1d-expressing), MHC-II-deficient (A^-/- or A^-/- KO) or MHC-I-deficient (homozygous transgenic mice deficient for ₂-microglobulin (₂m^-/-) or CD1d^-/-) environment into congenic, severely immunodeficient RAG1^-/- hosts to test their IBD-inducing potential. Unexpectedly, the adoptively transferred MHC-II-independent CD4⁺ T cells efficiently induced colitis. This observation points to a new feature of MHC-II-independent CD4⁺ T cells, their potential to trigger inflammatory Th1-type reactions in the mucosa.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Normal B6 mice, B6 MHC-II^-/- (A^-/- or A^-/-) mice (16, 17), C57BL/6J-Rag1^tm1Mom (RAG1^-/-) mice (39) (The Jackson Laboratory, Bar Harbor, ME), CD1d-deficient (CD1d^-/-) B6 mice (34) and B6 MHC-I^-/- (₂m^-/-) mice (40) were used. Mice were bred and kept under specific pathogen-free conditions in the animal facility of Ulm University. RAG1^-/- mice were transplanted at 8–12 wk of age.

Flow cytometry (FCM) analyses of the surface phenotype and intracellular cytokine expression

Cells were suspended in PBS/0.3% w/v BSA supplemented with 0.1% w/v sodium azide. Nonspecific binding of Abs to Fc receptor was blocked by preincubating cells with the mAb 2.4G2 directed against the FcRIII/II CD16/CD32 (1 µg mAb/10⁶ cells/100 µl). Cells were incubated with 0.5 µg/10⁶ cells of the relevant mAb for 30 min at 4°C and washed. In most experiments, cells were subsequently incubated with a second-step reagent for 10 min at 4°C. Three-color FCM analyses were performed on a FACSCalibur (BD Becton Dickinson, Mountain View, CA). The forward narrow angle light scatter was used as an additional parameter to facilitate exclusion of dead cells and aggregated cell clumps. The following reagents and mAb were obtained from BD PharMingen (Hamburg, Germany): PE-conjugated anti-CD3 mAb 145-2C11, FITC- and PE-conjugated anti-CD4 mAb GK1.5, biotinylated anti-CD4 mAb RM4-5, FITC-conjugated anti-CD8 mAb 53-6.7, biotinylated anti-CD44 (Pgp-1) mAb IM7, biotinylated anti-CD45RB mAb 23G2, biotinylated anti-CD28 mAb 37.51, biotinylated anti-CD69 mAb H1.2F3, PE-conjugated anti-NK1.1 mAb PK136, biotinylated anti-CD40L mAb MR1, and FITC-conjugated anti-CD62L (L-selectin) mAb MEL-14. PE-conjugated streptavidin was obtained from PharMingen. SA-Red670 was obtained from Life Technologies (Berlin, Germany).

Cells (10⁶ cells/ml) were stimulated with 50 ng/ml PMA and 500 ng/ml ionomycin in the presence of 10 µg/ml brefeldin A in RPMI 1640/10% FCS for 12 h at 37°C with 5% CO₂. Cells were harvested, washed twice in staining buffer (PBS without Mg²⁺/Ca²⁺, 0.3% w/v BSA, 0.1% w/v sodium azide), incubated (15 min, 4°C) with purified 2.4G2 Ab to block nonspecific binding of Ab to Fc receptors, washed with staining buffer, resuspended in staining buffer, and surface stained with the relevant Abs. Cells were washed with staining buffer, labeled with the second-step reagent, and washed twice. Cells were then resuspended in 100 µl Cytofix/Cytoperm solution for 20 min at 4°C and washed twice in 1 ml 1x Perm/Wash solution. Fixed and permeabilized cells were resuspended in 100 µl of 1x Perm/Wash solution. Cells were stained for 30 min at 4°C with 1 µg mAb/10⁶ cells of FITC-conjugated anti-IL-4 mAb BVD4-1D11, FITC-conjugated anti-IL-10 mAb JES5-16E3, FITC-conjugated anti-TNF- mAb MP6-XT22, FITC-conjugated anti-IFN- mAb XMG1.2, or appropriate negative control Abs (FITC-conjugated rat IgG1 mAb R3-34; PE-conjugated rat IgG1 mAb R3-34). Cells were washed twice in 1x Perm/Wash solution (250 x g) and resuspended in staining buffer; 10⁴ cells were analyzed by FCM using a FACScan equipped with a 15-mW argon laser (BD Becton Dickinson) using the CellQuest software (BD Becton Dickinson).

CD4⁺ T cells used for adoptive transfer

CD4⁺ T cells were aseptically purified from spleen cells depleted of CD8⁺ T cells by treatment with anti-CD8 Ab and low toxicity rabbit complement (Cedarlane Laboratories, Hornby, Ontario, Canada) following the manufacturer’s instructions. CD4⁺ T cells were enriched to >98% purity by positive selection on MACS separation columns (Milteny Biotec, Bergisch-Gladbach, Germany). Briefly, cell suspensions were washed in MACS buffer (PBS without Mg²⁺ and Ca²⁺ supplemented with 2 mM EDTA and 0.5% BSA) and incubated 20 min at 4°C with MACS CD4 MicroBeads. The magnetically labeled positive fraction was retained in a magnetic field on VS⁺ MACS columns. The purity of the positively separated CD4⁺ population was routinely >98%. Into RAG^-/- B6 mice, 3 x 10⁵ CD4⁺ cells were injected i.p. At biweekly intervals, the transplanted mice were weighed and their clinical condition was monitored.

Isolation of lymphoid cell populations from transplanted mice

Transplanted RAG^- mice were sacrificed by cervical dislocation. Single cell suspensions were aseptically prepared from the spleen, the mesenteric lymph nodes, and the lamina propria of the intestine. Colonic LPLs were isolated as described (12, 41, 42).

Generation of myeloid dendritic cells (DC) from bone marrow

The in vitro generation of myeloid DC from murine bone marrow has been described (43). Briefly, bone marrow cells prepared from femurs were depleted of CD4⁺CD8⁺B220⁺ lymphocytes and MHC-class-II⁺ cells (Miltenyi Biotec) by MACS sorting. These bone marrow cells depleted of T cells, B cells, and maturing myeloid cells were cultured at a density of 10⁶ cells/ml in 6-well plates (Nunc, Wiesbaden, Germany) in serum-free UltraCulture medium (BioWhittaker, Verviers, Belgium) supplemented with 5 ng/ml GM-CSF and 10 ng/ml FL (PeproTech, Rocky Hill, NJ), 2 mM glutamine, and antibiotics. Cultures were incubated at 37°C in humidified air supplemented with 5% CO₂. On days 3 and 5, cells were fed by medium exchange. From day 7 of cultures, nonadherent CD11c⁺ cells were purified by magnetic bead separation (Miltenyi Biotec), pulsed with bacterial lysates, and used as stimulator cells for CD4⁺ T cells.

Cytokine determination by ELISA

The release of cytokines by CD4⁺ T cells was detected by a conventional double-sandwich ELISA. For detection and capture, the mAb R4-6A2 and biotinylated mAb AN18 were used for IFN- (PharMingen). Extinction was analyzed at 405/490 nm on Spectra-Max equipment (Molecular Devices, Sunnyvale, CA) using the Softmax Pro software (Molecular Devices).

Histopathological examinations

Tissue samples were taken from various locations of the small and large intestine (duodenum, jejunum, terminal ileum, cecum, ascending colon, transverse colon, recto-sigmoid colon). Tissue was fixed in neutral buffered formalin, embedded in paraffin, sectioned on a microtome, mounted on slides, and stained with hematoxylin/eosin.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization of CD4⁺ T cells found in normal, MHC-II-deficient (A^-/- or A^-/-), or MHC-I-deficient (₂m^-/-, CD1d^-/-) B6 mice

We analyzed the surface phenotype, the inducible expression of proinflammatory cytokines, the response to -GalCer, and the diversity of the TCR V repertoire of CD4⁺ T cells from the B6 lines that were used as donors in the adoptive transfer experiments described below. Approximately one-third of the mononuclear cells from the spleen or cLP from normal, MHC-II-deficient (A^-/- or A^-/-) or MHC-I-deficient (₂m^-/- or CD1d^-/-) B6 mice were CD3⁺ T cells. As expected, A^-/- and A^-/- mice had few CD4⁺ T cells, and ₂m^-/- mice had few CD8⁺ T cells. NK1⁺CD4⁺ T cells were found in all mice tested. A 1–3% fraction of the splenic T cells from normal and MHC-I-deficient mice were NK1⁺, and the majority of splenic CD4⁺ T cells from A^-/- and A^-/- (MHC-II^-/-) B6 mice were NK1⁺. Furthermore, 5–15% of cLP CD4⁺ and CD8⁺ T cells from normal mice and the majority of cLP CD4⁺ T cell populations from A^-/- and A^-/- KO mice were NK1⁺. Within the splenic NK1⁺ CD3⁺ T cell populations in both normal and MHC-II-deficient B6 mice 30–45% were CD4⁺CD8^-, while the remaining NK1⁺ T cells were CD4^-CD8^- or CD4^-CD8⁺. Splenic NK1⁺CD4⁺ T cells showed an effector/memory T cell phenotype with high expression of costimulator molecules but also high CD45RB expression (data not shown).

Twice as many splenic CD4⁺ T cells from MHC-II-deficient than from normal B6 mice produced high levels of TNF- and/or IFN- after a 12-h incubation with phorbol ester and ionomycin (Fig. 1A). Splenic CD4⁺ T cells from normal and MHC-II-deficient (A^-/- or A^-/-) B6 mice proliferated and produced IFN- in response to stimulation by Con A, anti-CD3 Ab or -GalCer; splenic CD4⁺ T cells from MHC-I-deficient (₂m^-/-, CD1d^-/-) B6 mice responded to polyclonal stimulation by Con A or anti-CD3 Ab but not to stimulation by -GalCer (Fig. 1B, and data not shown). cLP CD4⁺ T cells from all lines released IFN- in response to polyclonal T cell stimulation by Con A or anti-CD3 Ab but did not respond to -GalCer stimulation (Fig. 1B, and data not shown). The TCR V repertoire of CD4⁺ T cells from the spleen and the cLP of normal B6 or MHC-II-deficient A^-/- KO B6 mice was diverse (data not shown).

View larger version (19K): [in this window] [in a new window]   FIGURE 1. A, Cytokine expression by splenic CD4+ T cells from normal(MHC-II-competent) or MHC-II-deficient B6 mice. Spleen cells from normal or A-/- KO B6 mice were stimulated for 10 h with phorbol ester/ionomycin and the fraction of cytoplasmic cytokine-expressing CD4+ T cells was determined by FACS analyses (data from three mice ± SEM are shown). B, IFN- release of splenic and cLP CD4+ T cells stimulated for 3 day with either immobilized anti-CD3 mAb 145-2C11 or -GalCer-pulsed CD1d+ J774 cells. CD4+ T cells were isolated from the spleen or the cLP of normal B6 mice (B6), MHC-II-deficient A-/- KO B6 mice (A-/-), MHC-I-deficient 2m-/- KO B6 mice (2m-/-), or CD1d-/- KO B6 mice (CD1d-/-). IFN- released into the supernatant was determined by ELISA (mean values ± SEM of triplicates is shown).

When splenic CD4⁺ T cells from either normal or MHC-II-deficient (A^-/- or A^-/- KO) B6 mice were injected into congenic RAG1^-/- B6 hosts (3 x 10⁵ cells/mouse), these mice showed diarrhea and rectal prolapse within 3–6 wk posttransfer (Fig. 2A). Many mice lost >20% of their body weight within 3–6 wk posttransfer. Loss in body weight did not strictly correlate with disease severity, because some transplanted mice died early posttransfer without significant loss of body weight, although they showed a histopathology of severe IBD. This IBD was progressive, severe, and always lethal. The course of the IBD in B6 RAG1^-/- hosts induced by the transfer of congenic CD4⁺ T cells was more aggressive and showed less interindividual variability than did the IBD in SCID mice induced by the transfer of BALB/c-derived CD4⁺ T cells that we have described in detail previously (12). Of particular interest was the observation that CD4⁺ T cells derived from MHC-II-deficient (A^-/- or A^-/-) B6 donor mice induced an aggressive form of the disease. Transfer of titrated numbers of purified CD4⁺ T cells from B6 A^-/- donor mice into RAG1^-/- hosts confirmed the efficient IBD-inducing potential of MHC-II-independent CD4⁺ T cells (Fig. 2B). CD4⁺ T cells from A^-/- and A^-/- KO B6 donor mice were equally efficient in inducing IBD (data not shown). These data reveal a new feature of MHC-II-independent CD4⁺ T cells: their unexpected efficiency in inducing mucosal inflammation.

View larger version (21K): [in this window] [in a new window]   FIGURE 2. RAG-/- mice transplanted with congenic CD4+ T cell populations develop IBD associated with weight loss. Left, RAG-/- B6 mice were transplanted with 3 x 105 purified splenic CD4+ T cells from normal B6 mice (B6), MHC-II-deficient A-/- KO B6 mice (A-/-), MHC-I-deficient 2m-/- KO B6 mice (2m-/-), or CD1d-/- KO B6 mice (CD1d-/-). Mean values (+ SEM) of 6–16 mice/group are shown. Right, RAG-/- B6 mice were transplanted with 106, 105, or 104 purified splenic CD4+ T cells from MHC-II-deficient A-/- KO B6 mice. Mean values (+SEM) of four mice/group are shown.

View larger version (90K): [in this window] [in a new window]   FIGURE 3. Histopathology of IBD (hematoxylin/eosin staining). Mice transplanted with CD4+ T cells from normal B6 donors (A and B) showed only a mild colitis at week 2 (A) with mucosal hyperplasia, a reduced number of goblet cells, and scanty inflammatory cells within the lamina propria. At week 4 (B), overt colitis with a dense inflammatory infiltrate, goblet cell depletion, and circumscribed epithelial erosions had developed. A more aggressive course of disease was observed when CD4+ T cells from B6 A-/- mice were transferred (C and D). Two weeks posttransfer (C), there was already a fully established colitis that further progressed to a very severe IBD by week 4 (D) characterized by an advanced disturbance of the cryptal architecture and extensive erosions of the mucosal epithelium. Animals that had received CD4+ T cells from B6 2m-/- donors (E and F) showed a normal colonic histology 2 wk after transplantation (E) and only a mild colitis in a minority of mice (5/22) at week 4 (F). Arrowheads in B, C, and D indicate epithelial erosions. Magnifications in A–F: x36.

We isolated cLP T cells from RAG1^-/- mice with severe IBD that were transplanted with splenic CD4⁺ T cells from MHC-II-dependent or -independent B6 donor mice. The phenotype of IBD-associated cLP CD4⁺ T cells indicated that they are activated in situ evident by low expression of CD62L and high expression of CD69 and CD44 in all cLP CD4⁺ T cell populations tested. A fraction (20–30%) of cLP CD4⁺ T cells from RAG1^-/- hosts reconstituted with CD4⁺ T cells from normal or A^-/- B6 donor mice was NK1⁺. Surface expression of NK1.1 by cLP CD4⁺ T cells was lower than by cLP NK cells. In FCM analyses, we characterized the surface phenotype of the NK1⁺ and NK1^- subsets of the cLP CD4⁺ T cell populations because differences were apparent in the surface phenotype of cLP NK1⁺ vs NK1^- CD4⁺ T cells (Fig. 4). cLP NK1⁺ CD4⁺ T cells from diseased RAG^-/- hosts expressed higher surface levels of CD45RB than did NK1^- CD4⁺ T cell in the same LPL population. Surface expression of CD28 was higher in NK1⁺ CD4⁺ T cells than in NK1^- CD4⁺ T cells. Expression of CD40L was low in NK1⁺ and NK1^- CD4⁺ T cells.

View larger version (49K): [in this window] [in a new window]   FIGURE 4. Surface phenotype of cLP NK1+ and NK1-CD4+ T cells from transplanted RAG-/- hosts with colitis. cLP CD4+ T cells were obtained from RAG-/- hosts with IBD transplanted 20 days previously with CD4+ T cells from either normal (MHC-II+) B6 mice or MHC-II-deficient (MHC-II-) A-/- KO B6 mice. Cells were labeled and analyzed by three-color FCM.

View larger version (35K): [in this window] [in a new window]   FIGURE 5. Cytokine expression by cLP CD4+ T cells from RAG-/- mice with colitis that were transplanted with CD4+ T cells from either normal (MHC-II+) B6 mice or MHC-II-deficient (MHC-II-) A-/- KO B6 mice. cLP CD4+ T cells were obtained from RAG-/- hosts with IBD transplanted 20 days previously with CD4+ T cells. These T cells were stimulated for 6 h with PMA/Iono. Surface and cytoplasmic cytokine expression was analyzed by FCM.

View larger version (25K): [in this window] [in a new window]   FIGURE 6. IFN- released by IBD-associated, cLP CD4+ T cells from (A) normal (MHC-II-competent, A+/+) or (B) MHC-II-deficient (A-/-) donor mice stimulated by bacterial lysate-pulsed DC. Myeloid DC were grown in serum-free cultures from bone marrow cells of normal B6 donor mice (A and B, upper group), MHC-II-deficient (A-/-) KO B6 donor mice (B), or MHC-I-deficient (2m-/-, CD1d-/-) KO B6 mice (B). DC were pulsed with bacterial lysates for 12 h, washed extensively, and used as stimulator cells in cocultures with purified cLP CD4+ T responder cells (at a ratio of 1:5). After a 48-h coculture, released IFN- was determined in the supernatant by ELISA. In some cultures 10 µg/ml of the anti-Ab mAb M5/114.15.2 was added. The values are mean pg/ml IFN- ± SEM of triplicates.

MHC-II-independent CD4⁺ T cells efficiently induced a colitis after transfer into RAG1^-/- hosts. CD1d-restricted and -GalCer-reactive NKT cells are the only well-characterized, MHC-II-independent CD4⁺ T cell subset (27, 30, 46, 47). In cLP CD4⁺ T cell populations, we found NK1⁺ T cells. cLP T cells from transplanted and diseased hosts did not respond to -GalCer-pulsed CD1d⁺ DC (data not shown). This indicated that well-characterized CD1d-restricted NK1⁺ CD4⁺ T cells reactive to -GalCer are not involved in IBD induction. This was confirmed in transfer experiments using CD4⁺ T cells from CD1d^-/- KO B6 donor mice. RAG1^-/- B6 hosts injected with purified, splenic CD4⁺ T cells from CD1d^-/- KO B6 mice developed a colitis (Fig. 2). The time course of this disease, its histopathology, and the phenotype of the cLP CD4⁺ T cells isolated from diseased RAG1^-/- resembled the IBD induced by transfer of normal B6 CD4⁺ T cells (data not shown). Furthermore, transfer of CD4⁺ T cells from J281^-/- KO B6 donor mice into RAG1^-/- B6 mice induced a colitis with a clinical course and histopathology similar to that induced by normal B6 CD4⁺ T cells (data not shown). cLP CD4⁺ T cells stimulated with lysate-pulsed DC from CD1d^-/- B6 KO mice released IFN- indicating that this MHC-I-like molecule is not involved (data not shown). In the last set of experiments, we transferred CD4⁺ T cells from MHC-I-deficient ₂m^-/- KO B6 mice into RAG1^-/- hosts to test whether MHC-I-dependent CD4⁺ T cells are required to drive the disease process.

CD4⁺ T cells from MHC-I-deficient ₂m^-/- KO B6 donors induce no (or only very mild) colitis in RAG1^-/- KO hosts

CD4⁺ T cells were found in the spleen and cLP of MHC-I-deficient ₂m^-/- KO B6 mice. After transfer into RAG1^-/- hosts, these cells induced no or only mild histopathological signs of colitis during a 10- to 16-wk observation period that were not accompanied by clinical signs (Fig. 2A). Signs of a mild colitis were found only in 5/22 RAG1^-/- hosts transplanted with purified CD4⁺ T cells from ₂m^-/- KO B6 donor mice (Fig. 3F); the other hosts showed an essentially normal histology without signs of inflammation (Fig. 3A). The surface phenotype of cLP CD4⁺ from RAG1^-/- mice transplanted with T cells from ₂m^-/- KO B6 donors was similar to that described above for cLP CD4⁺ T cells from RAG1^-/- hosts transplanted with T cells from normal, MHC-II-deficient or CD1d-deficient B6 donor mice (Fig. 7A). Striking differences were observed in the cytokine expression profile of cLP CD4⁺ T cells from nondiseased RAG1^-/- hosts transplanted with CD4⁺ T cells from MHC-I-deficient ₂m^-/- KO B6 mice. Many of these T cells expressed IL-4 but few produced IFN- or TNF- (Fig. 7B). This Th2-biased cytokine expression pattern is in contrast to the Th1-biased pattern described above for cLP CD4⁺ T cells from RAG1^-/- B6 mice with colitis transplanted with CD4⁺ T cells from normal B6, MHC-II-deficient (A^-/- or A^-/-) B6, or CD1d^-/- KO B6 mice. These data point to MHC-I/₂m-dependent (but not CD1d-restricted) CD4⁺ T cells that promote the Th1-biased polarization of mucosal T cell responses in the colon.

View larger version (44K): [in this window] [in a new window]   FIGURE 7. Surface phenotype and cytokine expression by cLP CD4+ T cells from 2m-/- KO B6 donor mice engrafted into RAG-/- hosts that showed no evidence of colitis. cLP CD4+ T cells were obtained from RAG-/- hosts transplanted 8 wk previously with CD4+ T cells from 2m-/- KO B6 donor mice. Cells were labeled and their surface phenotype was analyzed by three-color FCM. These T cells were stimulated for 6 h with PMA/Iono, and their surface and cytoplasmic cytokine expression was analyzed.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The main finding of our study was the unexpected potency of MHC-II-independent CD4⁺ T cells to induce colitis. CD4⁺ T cells from either A^-/- or A^-/- KO B6 mice efficiently induced a colitis in congenic immunodeficient RAG1^-/- B6 hosts. This excludes the possibility that minor populations of MHC-II (AE)-dependent CD4⁺ T cells from A^-/- KO mice can induce colitis or that minor histocompatibility differences between donor and host play a role (A^-/- KO mice were generated in B6 mice; A^-/- KO mice were from the 14th backcross to B6). The MHC-II-independent CD4⁺ T cell subset that induced colitis is unknown. These T cells are present in normal, MHC-II^-/- (A^-/- or A^-/-), CD1d^-/-, J281^-/- but not in MHC-I^-/- (₂m^-/-) B6 mice. They are inducible to rapidly express proinflammatory cytokines (TNF-, IFN-), and they repopulate and expand in the immunodeficient host. IBD-associated cLP CD4⁺ T cells from A^-/- or A^-/- KO B6 donor mice showed variable NK1 expression, did not respond to -GalCer, and showed (in most but not all mice) an oligoclonal TCR V5 bias. MHC-II-independent CD4⁺ T cells involved in inducing IBD are not the well-characterized CD4⁺ NKT cells. NKT cells are a fairly constant T cell subset of 10⁶ cells/organ in many lymphoid and nonlymphoid organs of the mouse. The majority of CD4⁺ NKT cells are in vivo activated, rapidly release cytokines after stimulation, express intermediate levels of CD3 and TCR, and use an invariant V14J281 TCR -chain (with an invariant CDR3 region containing no N-region additions/deletions) paired with V8.2, V7, or V2 (but not V5) TCR -chains (with a variable CDR3 region). CD4⁺ NKT cells recognize -GalCer (25, 26, 27, 28, 30) in the context of CD1d (17, 24, 36). In contrast to the restricted TCR V and V repertoire of CD1d-restricted, -GalCer-specific CD4⁺ NKT cells, other MHC-II-independent CD4⁺ T cells show a diverse TCR repertoire and do not recognize -GalCer. CD4⁺ T cells from A^-/- and A^-/- B6 donor mice that repopulated spleen, mesenteric lymph nodes, and gut mucosa of RAG1^-/- hosts showed a diverse TCR V repertoire at an early stage of the disease, but a restricted TCR V usage pattern as the disease progressed. In 3 of 5 late-stage, transplanted IBD⁺ mice that we analyzed in detail, >80% of the T cells expressed TCR V5. This was not observed in splenic or cLP CD4⁺ T cells isolated from 8 RAG1^-/- hosts transplanted with CD4⁺ T cells from normal, CD1d^-/- or J281^-/- KO mice (data not shown). MHC-II-independent CD4⁺ T cell populations that expand in the immunodeficient host during the emergence of IBD, thus, tend to become oligoclonal. This suggests that a selection process drives mucosal repopulation.

Histopathological evidence of colitis was observed in only 5 of 22 RAG1^-/- mice transplanted with CD4⁺ T cells from ₂m^-/- KO B6 mice (in four independent experiments). Histologically, these five animals showed only mild colitis late in a 10- to 16-wk observation period. The surface phenotype of cLP CD4⁺ T cells from these mice did not differ from that of cLP CD4⁺ T cells from severely diseased RAG1^-/- mice transplanted with CD4⁺ T cells from normal B6, A^-/- KO or A^-/- KO B6 mice (Fig. 7A). The only striking difference observed was the cytokine expression profile of cLP CD4⁺ T cells from transplanted RAG1^-/- mice. Although cLP CD4⁺ T cells from diseased RAG1^-/- mice transplanted with T cells from normal, A^-/- or A^-/- B6 mice produced TNF- and IFN- but no IL-4, only few cLP CD4⁺ T cells derived from ₂m^-/- KO B6 mice produced TNF- or IFN- but many produced IL-4 and IL-10 (Fig. 7B). The lack or very mild course of colitis, thus, coincided with a Th2-biased polarization of the T cell response in the cLP. A control of aggressive CD4⁺ T cells (derived from CD45RB^high precursors) by suppressive CD4⁺ T cells (derived from CD45RB^low precursors) in the pathogenesis of colitis has been proposed (3, 11, 51, 52, 53, 54). A predominance of suppressive CD4⁺ T cells in ₂m^-/- B6 mice may explain the low incidence of colitis induction. When MHC-II-independent CD4⁺ T cells (from A^-/- B6 mice) were either mixed to MHC-I-independent CD4⁺ T cells (from ₂m^-/- B6 mice) or transferred into disease-free RAG^-/- hosts repopulated for 8–14 wk with CD4⁺ T cells from ₂m^-/- donor B6 mice, a colitis developed (unpublished data). This argues against a dominant suppressive activity of CD4⁺ T cells from ₂m^-/- donor mice. It has been shown that IL-2^-/- x ₂m^-/- double KO mice spontaneously develop a CD4⁺ T cell-dependent colitis (55). In contrast to our data, MHC-II-dependent but not MHC-I-dependent CD4⁺ T cells are critical for the development of colitis in this model. It has been reported that the transfer of 4–5 x 10⁵ CD45RB^high CD4⁺ T cells into MHC-II^-/- x RAG^-/- double KO hosts does not induce colitis (56). This graft is expected to contain 10³ MHC-II-independent CD4⁺ T cells. In the absence of MHC-II-dependent CD4⁺ T cells, this small number of transferred MHC-I-dependent CD4⁺ T cells, thus, does not induce colitis. Alternatively, MHC-I-dependent CD4⁺ T cells in A^-/- or A^-/- KO mice and in normal mice may differ.

We reported: 1) the efficient induction of IBD by CD4⁺ T cells from either normal, CD1d^-/- and J281^-/- mice, or MHC-II-deficient (A^-/- or A^-/-) mice; and 2) no (or inefficient) induction of IBD by CD4⁺ T cells from MHC-I-deficient (₂m^-/-) mice. Therefore, dysregulation of T cell reactivity seems to be an important initiating event in the pathogenesis of colitis. We described three experimental conditions in which MHC-I- and/or MHC-II-dependent CD4⁺ T cells were either present or absent (1). Transfer of CD4⁺ T cells from A^-/- or A^-/- KO mice: in the absence of conventional MHC-II-restricted CD4⁺ T cells, large numbers of MHC-II-independent CD4⁺ T cells expand and induce IBD (2). Transfer of CD4⁺ T cells from normal, CD1d^-/- or J281^-/- mice: in the presence of low numbers of MHC-II-independent CD4⁺ T cells and large numbers of conventional MHC-II-restricted CD4⁺ T cells, MHC-II-dependent CD4⁺ T cells expand polyclonally, develop a Th1-biased reactivity, and induce IBD (3). Transfer of CD4⁺ T cells from ₂m^-/- mice: only conventional MHC-II-restricted CD4⁺ T cells expand polyclonally that do not induce IBD because they default to a Th2-biased reactivity (in the absence of regulatory MHC-II-independent CD4⁺ T cells). MHC-I-dependent CD4⁺ T cells, thus, seem to facilitate Th1-biased, mucosal CD4⁺ T cell responses. A regulatory role of NKT cells has been proposed in the pathogenesis of graft-versus-host disease and autoimmune disease (57, 58, 59). To our knowledge, we provide the first evidence that MHC-II- and CD1d-independent CD4⁺ T cells can modulate the polarization of mucosal T cell responses in the gut.

	Acknowledgments

 
We thank Anja Müller for the expert technical assistance. We thank Drs. Mogens Claesson and Jens Brimnes (University of Copenhagen, Denmark) for the help and constructive discussion with bacterial lysate-pulsed DC cultures, and Dr. M. Kronenberg for the CD1d⁺ J774 cells and the anti-CD1d mAb.

	Footnotes

 
¹ This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (DFG Re549/9-1; to J.R.) and the IZKF/A7 (University of Ulm; to F.L.).

² Address correspondence and reprint requests to Dr. Jörg Reimann, Department of Medical Microbiology and Immunology, University of Ulm, Helmholtzstrasse, 8/1, D-89081, Ulm, Germany.

³ Abbreviations used in this paper: IBD, inflammatory bowel disease; cLP, colonic lamina propria; KO, knockout; NKT cells, T cells expressing the NK1 marker; -GalCer, -galactosyl ceramide; LPL, lamina propria lymphocyte; ₂m^-/-, homozygous transgenic mice deficient for ₂-microglobulin; FCM, flow cytometry; DC, dendritic cell(s); B6, C57BL/6.

Received for publication August 22, 2000. Accepted for publication January 3, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Morrissey, P. J., K. Charrier, S. Braddy, D. Liggitt, J. D. Watson. 1993. CD4⁺ T cells that express high levels of CD45RB induce wasting disease when transferred into congenic severe combined immunodeficient mice: disease development is prevented by cotransfer of purified CD4⁺ T cells. J. Exp. Med. 178:237.[Abstract/Free Full Text]
2. Powrie, F., M. W. Leach, S. Mauze, S. Menon, L. Barcomb-Caddle, R. L. Coffman. 1994. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RB^hi T cells. Immunity 1:553.[Medline]
3. Powrie, F., R. Correa Oliveira, S. Mauze, R. L. Coffman. 1994. Regulatory interactions between CD45RB^high and CD45RB^low CD4⁺ T cells are important for the balance between protective and pathogenic cell-mediated immunity. J. Exp. Med. 179:589.[Abstract/Free Full Text]
4. Rudolphi, A., G. Boll, S. S. Poulsen, M. H. Claesson, J. Reimann. 1994. Gut-homing CD4⁺ TCR⁺ T cells in the pathogenesis of murine inflammatory bowel disease. Eur. J. Immunol. 24:2803.[Medline]
5. Aranda, R., B. C. Sydora, P. L. McAllister, S. W. Binder, H. Y. Yang, S. R. Targan, M. Kronenberg. 1997. Analysis of intestinal lymphocytes in mouse colitis mediated by transfer of CD4⁺ CD45RB^hi T cells to SCID recipients. J. Immunol. 158:3464.[Abstract]
6. Simpson, S. J., S. Shah, M. Comiskey, J. Y. de, B. Wang, E. Mizoguchi, A. K. Bhan, C. Terhorst. 1998. T cell-mediated pathology in two models of experimental colitis depends predominantly on the interleukin12/signal transducer and activator of transcription (Stat)-4 pathway, but is not conditional on interferon- expression by T cells. J. Exp. Med. 187:1225.[Abstract/Free Full Text]
7. Corazza, N., S. Eichenberger, H.-P. Eugster, C. Mueller. 1999. Nonlymphocyte-derived tumor necrosis factor is required for induction of colitis in recombinant activating gene RAG2^-/- mice upon transfer of CD4⁺ CD45RB^hi T cells. J. Exp. Med. 190:1479.[Abstract/Free Full Text]
8. Annacker, O., O. Burlen-Defranoux, R. Pimenta-Araujo, A. Cumano, A. Bandeira. 2000. Regulatory CD4 T cells control the size of the peripheral activated/memory CD4 T cell compartment. J. Immunol. 164:3573.[Abstract/Free Full Text]
9. De-Winter, H., H. Cheroutre, M. Kronenberg. 1999. Mucosal immunity and inflammation: the yin and yang of T cells in intestinal inflammation: pathogenic and protective roles in a mouse colitis model. Am. J. Physiol. 276:G1317.[Abstract/Free Full Text]
10. Strober, W., B. R. Ludviksson, I. J. Fuss. 1998. The pathogenesis of mucosal inflammation in murine models of inflammatory bowel disease and Crohn disease. Ann. Intern. Med. 128:848.[Abstract/Free Full Text]
11. Powrie, F.. 1995. T cells in inflammatory bowel disease: protective and pathogenic roles. Immunity 3:171.[Medline]
12. Claesson, M. H., S. Bregenholt, K. Bonhagen, S. Thoma, P. Moller, M. J. Grusby, F. Leithauser, M. H. Nissen, J. Reimann. 1999. Colitis-inducing potency of CD4⁺ T cells in immunodeficient, adoptive hosts depends on their state of activation, IL-12 responsiveness, and CD45RB surface phenotype. J. Immunol. 162:3702.[Abstract/Free Full Text]
13. Simpson, S. J., G. A. Hollander, E. Mizoguchi, D. Allen, A. K. Bhan, B. Wang, C. Terhorst. 1997. Expression of pro-inflammatory cytokines by TCR⁺ and TCR⁺ T cells in an experimental model of colitis. Eur. J. Immunol. 27:17.[Medline]
14. Steinhoff, U., V. Brinkmann, U. Klemm, P. Aichele, P. Seiler, U. Brandt, P. W. Bland, I. Prinz, U. Zugel, S. H. Kaufmann. 1999. Autoimmune intestinal pathology induced by hsp60-specific CD8 T cells. Immunity 11:349.[Medline]
15. Mombaerts, P., E. Mizoguchi, M. J. Grusby, L. H. Glimcher, A. K. Bhan, S. Tonegawa. 1993. Spontaneous development of inflammatory bowel disease in T cell receptor mutant mice. Cell 75:275.
16. Kontgen, F., G. Suss, C. Stewart, M. Steinmetz, H. Bluethmann. 1993. Targeted disruption of the MHC class II A gene in C57.BL/6 mice. Int. Immunol. 5:957.[Abstract/Free Full Text]
17. Cardell, S., S. Tangri, S. Chan, M. Kronenberg, C. Benoist, D. Mathis. 1995. CD1-restricted CD4⁺ T cells in major histocompatibility complex class II-deficient mice. J. Exp. Med. 182:993.[Abstract/Free Full Text]
18. Lee, D. J., A. Abeyratne, D. A. Carson, M. Corr. 1998. Induction of an antigen-specific, CD1-restricted cytotoxic T lymphocyte response in vivo. J. Exp. Med. 187:433.[Abstract/Free Full Text]
19. Park, S. H., J. H. Roark, A. Bendelac. 1998. Tissue-specific recognition of mouse CD1 molecules. J. Immunol. 160:3128.[Abstract/Free Full Text]
20. Tangri, S., L. Brossay, N. Burdin, D. J. Lee, M. Corr, M. Kronenberg. 1998. Presentation of peptide antigens by mouse CD1 requires endosomal localization and protein antigen processing. Proc. Natl. Acad. Sci. USA 95:14314.[Abstract/Free Full Text]
21. Schofield, L., M. J. McConville, D. Hansen, A. S. Campbell, R. B. Fraser, M. J. Grusby, S. D. Tachado. 1999. CD1.d-restricted immunoglobulin G formation to GPI-anchored antigens mediated by NKT cells. Science 283:225.[Abstract/Free Full Text]
22. MacDonald, H. R.. 2000. CD1d-glycolipid tetramers: a new tool to monitor natural killer T cells in health and disease. J. Exp. Med. 192:F15.[Medline]
23. MacDonald, H. R.. 1995. NK1.1⁺ T cell receptor-⁺ cells: new clues to their origin, specificity, and function. J. Exp. Med. 182:633.[Free Full Text]
24. Bendelac, A., M. N. Rivera, S. H. Park, J. H. Roark. 1997. Mouse CD1-specific NK1 T cells: development, specificity, and function. Annu. Rev. Immunol. 15:535.[Medline]
25. Kawano, T., J. Cui, Y. Koezuka, I. Toura, Y. Kaneko, K. Motoki, H. Ueno, R. Nakagawa, H. Sato, E. Kondo, et al 1997. CD1.d-restricted and TCR-mediated activation of V14 NKT cells by glycosylceramides. Science 278:1626.[Abstract/Free Full Text]
26. Brossay, L., O. V. Naidenko, N. Burdin, J. L. Matsuda, T. Sakai, M. Kronenberg. 1998. Structural requirements for galactosylceramide recognition by CD1-restricted NK T cells. J. Immunol. 161:5124.[Abstract/Free Full Text]
27. Brossay, L., M. Chioda, N. Burdin, Y. Koezuka, G. Casorati, P. Dellabona, M. Kronenberg. 1998. CD1.d-mediated recognition of an -galactosylceramide by natural killer T cells is highly conserved through mammalian evolution. J. Exp. Med. 188:1521.[Abstract/Free Full Text]
28. Burdin, N., L. Brossay, Y. Koezuka, S. T. Smiley, M. J. Grusby, M. Gui, M. Taniguchi, K. Hayakawa, M. Kronenberg. 1998. Selective ability of mouse CD1 to present glycolipids: -galactosylceramide specifically stimulates V14⁺ NK T lymphocytes. J. Immunol. 161:3271.[Abstract/Free Full Text]
29. Burdin, N., L. Brossay, M. Kronenberg. 1999. Immunization with -galactosylceramide polarizes CD1-reactive NK T cells towards Th2 cytokine synthesis. Eur. J. Immunol. 29:2014.[Medline]
30. Kitamura, H., K. Iwakabe, T. Yahata, S. Nishimura, A. Ohta, Y. Ohmi, M. Sato, K. Takeda, K. Okumura, L. Van-Kaer, et al 1999. The natural killer T (NKT) cell ligand -galactosylceramide demonstrates its immunopotentiating effect by inducing interleukin (IL)-12 production by dendritic cells and IL-12 receptor expression on NKT cells. J. Exp. Med. 189:1121.[Abstract/Free Full Text]
31. Singh, N., S. Hong, D. C. Scherer, I. Serizawa, N. Burdin, M. Kronenberg, Y. Koezuka, L. Van-Kaer. 1999. Activation of NK T cells by CD1.d and -galactosylceramide directs conventional T cells to the acquisition of a Th2 phenotype. J. Immunol. 163:2373.[Abstract/Free Full Text]
32. Chen, Y. H., N. M. Chiu, M. Mandal, N. Wang, C.-R. Wang. 1997. Impaired NK1⁺ T cell development and early IL-4 production in CD1-deficient mice. Immunity 6:459.[Medline]
33. Mendiratta, S. K., W. D. Martin, S. Hong, A. Boesteanu, S. Joyce, L. Van-Kaer. 1997. CD1.d1 mutant mice are deficient in natural T cells that promptly produce IL-4. Immunity 6:469.[Medline]
34. Smiley, S. T., M. H. Kaplan, M. J. Grusby. 1997. Immunoglobulin E production in the absence of interleukin-4-secreting CD1-dependent cells. Science 275:977.[Abstract/Free Full Text]
35. Guy-Grand, D., J. B. Cuenod, S. M. Malassis, F. Selz, P. Vassalli. 1996. Complexity of the mouse gut T cell immune system: identification of two distinct natural killer T cell intraepithelial lineages. Eur. J. Immunol. 26:2248.[Medline]
36. Ohteki, T., H. R. MacDonald. 1994. Major histocompatibility complex class I related molecules control the development of CD4⁺8^- and CD4^-8^- subsets of natural killer 1.1⁺ T cell receptor-⁺ cells in the liver of mice. J. Exp. Med. 180:699.[Abstract/Free Full Text]
37. Benlagha, K., A. Weiss, A. Beavis, L. Teyton, A. Bendelac. 2000. In vivo identification of glycolipid antigen-specific T cells using fluorescent CD1d tetramers. J. Exp. Med. 191:1895.[Abstract/Free Full Text]
38. Matsuda, J. L., O. V. Naidenko, L. Gapin, T. Nakayama, M. Taniguchi, C.-R. Wang, Y. Koezuka, M. Kronenberg. 2000. Tracking the response of natural killer T cells to a glycolipid antigen using CD1.d tetramers. J. Exp. Med. 192:741.[Abstract/Free Full Text]
39. Mombaerts, P., J. Iacomini, R. S. Johnson, K. Herrup, S. Tonegawa, V. E. Papaioannou. 1992. RAG-1-deficient mice have no mature B and T lymphocytes. Cell 68:869.[Medline]
40. Koller, B. H., P. Marrack, J. W. Kappler, O. Smithies. 1990. Normal development of mice deficient in ₂m, MHC class I proteins, and CD8⁺ T cells. Science 248:1227.[Abstract/Free Full Text]
41. Bonhagen, K., S. Thoma, P. Bland, S. Bregenholt, A. Rudolphi, M. H. Claesson, J. Reimann. 1996. Cytotoxic reactivity of gut lamina propria CD4⁺ T cells in SCID mice with colitis. Eur. J. Immunol. 26:3074.[Medline]
42. Bonhagen, K., S. Thoma, F. Leithauser, P. Moller, J. Reimann. 1998. A pancolitis resembling human ulcerative colitis (UC) is induced by CD4⁺ TCR T cells of athymic origin in histocompatible severe combined immunodeficient (SCID) mice. Clin. Exp. Immunol. 112:443.[Medline]
43. 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]
44. Cong, Y., S. L. Brandwein, R. P. McCabe, A. Lazenby, E. H. Birkenmeier, J. P. Sundberg, C. O. Elson. 1998. CD4⁺ T cells reactive to enteric bacterial antigens in spontaneously colitic C3H/HeJBir mice: increased T helper cell type 1 response and ability to transfer disease. J. Exp. Med. 187:855.[Abstract/Free Full Text]
45. Brimnes, J., J. Reimann, M. H. Nissen, and M. H. Claesson. 2000. Enteric bacterial antigens stimulate colonic lamina propria CD4⁺ T-cells in scid mice with inflammatory bowel disease. Eur. J. Immunol. In press.
46. Gumperz, J. E., C. Roy, A. Makowska, D. Lum, M. Sugita, T. Podrebarac, Y. Koezuka, S. A. Porcelli, S. Cardell, M. B. Brenner, et al 2000. Murine CD1.d-restricted T cell recognition of cellular lipids. Immunity 12:211.[Medline]
47. Grant, E. P., M. Degano, J. P. Rosat, S. Stenger, R. L. Modlin, I. A. Wilson, S. A. Porcelli, M. B. Brenner. 1999. Molecular recognition of lipid antigens by T cell receptors. J. Exp. Med. 189:195.[Abstract/Free Full Text]
48. Chen, H., H. Huang, W. E. Paul. 1997. NK1.1⁺ CD4⁺ T cells lose NK1.1 expression upon in vitro activation. J. Immunol. 158:5112.[Abstract]
49. Slifka, M. K., R. R. Pagarigan, J. L. Whitton. 2000. NK markers are expressed on a high percentage of virus-specific CD8⁺ and CD4⁺ T cells. J. Immunol. 164:2009.[Abstract/Free Full Text]
50. Assarsson, E., T. Kambayashi, J. K. Sandberg, S. Hong, M. Taniguchi, L. Van Kaer, H.-G. Ljunggren, B. J. Chambers. 2000. CD8⁺ T cells rapidly acquire NK1.1 and NK cell-associated molecules upon stimulation in vitro and in vivo. J. Immunol. 165:3673.[Abstract/Free Full Text]
51. Powrie, F., M. W. Leach, S. Mauze, L. B. Caddle, R. L. Coffman. 1993. Phenotypically distinct subsets of CD4⁺ T cells induce or protect from chronic intestinal inflammation in C. B-17 scid mice. Int. Immunol. 5:1461.[Abstract/Free Full Text]
52. Morrissey, P. J., K. Charrier. 1994. Induction of wasting disease in SCID mice by the transfer of normal CD4⁺/CD45RB^hi T cells and the regulation of this autoreactivity by CD4⁺/CD45RB^lo T cells. Res. Immunol. 145:357.[Medline]
53. Powrie, F., J. Carlino, M. W. Leach, S. Mauze, R. L. Coffman. 1996. A critical role for transforming growth factor- but not interleukin4 in the suppression of T helper type 1-mediated colitis by CD45RB^low CD4⁺ T cells. J. Exp. Med. 183:2669.[Abstract/Free Full Text]
54. Read, S., S. Mauze, C. Asseman, A. Bean, R. Coffman, F. Powrie. 1998. CD38⁺ CD45RB^lo CD4⁺ T cells: a population of T cells with immune regulatory activities in vitro. Eur. J. Immunol. 28:3435.[Medline]
55. Simpson, S. J., E. Mizoguchi, D. Allen, A. K. Bhan, C. Terhorst. 1995. Evidence that CD4⁺, but not CD8⁺ T cells are responsible for murine interleukin-2-deficient colitis. Eur. J. Immunol. 25:2618.[Medline]
56. Matsuda, J. L., L. Gapin, B. C. Sydora, F. Byrne, S. Binder, M. Kronenberg, R. Aranda. 2000. Systemic activation and antigen-driven oligoclonal expansion of T cells in a mouse model of colitis. J. Immunol. 164:2797.[Abstract/Free Full Text]
57. Lehuen, A., O. Lantz, L. Beaudoin, V. Laloux, C. Carnaud, A. Bendelac, J. F. Bach, R. C. Monteiro. 1998. Overexpression of natural killer T cells protects V14-J281 transgenic nonobese diabetic mice against diabetes. J. Exp. Med. 188:1831.[Abstract/Free Full Text]
58. Zeng, D., D. Lewis, J. S. Dejbakhsh, F. Lan, O. M. Garcia, R. Sibley, S. Strober. 1999. Bone marrow NK1.1^- and NK1.1⁺ T cells reciprocally regulate acute graft versus host disease. J. Exp. Med. 189:1073.[Abstract/Free Full Text]
59. Zeng, D., M. K. Lee, J. Tung, A. Brendolan, S. Strober. 2000. A role for CD1 in the pathogenesis of lupus in NZB/NZW mice. J. Immunol. 164:5000.[Abstract/Free Full Text]

This article has been cited by other articles:

				P. Bochtler, C. Wahl, R. Schirmbeck, and J. Reimann Functional Adaptive CD4 Foxp3 T Cells Develop in MHC Class II-Deficient Mice J. Immunol., December 15, 2006; 177(12): 8307 - 8314. [Abstract] [Full Text] [PDF]

				B. Bienvenu, B. Martin, C. Auffray, C. Cordier, C. Becourt, and B. Lucas Peripheral CD8+CD25+ T Lymphocytes from MHC Class II-Deficient Mice Exhibit Regulatory Activity J. Immunol., July 1, 2005; 175(1): 246 - 253. [Abstract] [Full Text] [PDF]

				K. Takeshita, T. Yamasaki, S. Akira, F. Gantner, and K. B. Bacon Essential role of MHC II-independent CD4+ T cells, IL-4 and STAT6 in contact hypersensitivity induced by fluorescein isothiocyanate in the mouse Int. Immunol., May 1, 2004; 16(5): 685 - 695. [Abstract] [Full Text] [PDF]

				L. H. Boyle and J. S. Hill Gaston Breaking the rules: the unconventional recognition of HLA-B27 by CD4+ T lymphocytes as an insight into the pathogenesis of the spondyloarthropathies Rheumatology, March 1, 2003; 42(3): 404 - 412. [Abstract] [Full Text] [PDF]

				M. J. Pinkoski, N. M. Droin, and D. R. Green Tumor Necrosis Factor alpha Up-regulates Non-lymphoid Fas-ligand following Superantigen-induced Peripheral Lymphocyte Activation J. Biol. Chem., October 25, 2002; 277(44): 42380 - 42385. [Abstract] [Full Text] [PDF]

				L. H. Boyle, J. C. Goodall, S. S. Opat, and J. S. H. Gaston The Recognition of HLA-B27 by Human CD4+ T Lymphocytes J. Immunol., September 1, 2001; 167(5): 2619 - 2624. [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 Trobonjaca, Z. Articles by Reimann, J. Search for Related Content PubMed PubMed Citation Articles by Trobonjaca, Z. Articles by Reimann, J.