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Originally published online as doi:10.1189/jlb.0703351 on October 13, 2003

Published online before print October 13, 2003
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(Journal of Leukocyte Biology. 2004;75:76-86.)
© 2004 by Society for Leukocyte Biology

Adoptive transfer of ex vivo immune-programmed NKT lymphocytes alleviates immune-mediated colitis

Oren Shibolet*,1, Yossef Kalish*,1, Athalia Klein*, Ruslana Alper*, Lydia Zolotarov*, Barbara Thalenfeld{dagger}, Dean Engelhardt{dagger}, Elazar Rabbani{dagger} and Yaron Ilan*,2

* Liver Unit, Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem, Israel; and
{dagger} ENZO Biochem, New York, New York

2Correspondence: Liver Unit, Department of Medicine, Hadassah University Hospital, P.O. Box 12000, Jerusalem, Israel IL-91120. E-mail: ilan{at}hadassah.org.il


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ABSTRACT
 
T lymphocyte-expressing natural killer (NK) cell markers (NKT cells) play a role in immune regulation. Our aim was to evaluate the in vivo effect of adoptive transfer of immune-programmed NKT cells. Colitis was induced in C57/B6 mice by 2,4,6-trinitrobenzenesulfonic acid. NKT, CD4, CD8 lymphocytes, and dendritic cells (DC) were prepared from spleens of naive mice, animals with colitis, and animals with colitis that were orally tolerized. Subsets of splenocytes, NKT, CD4, and CD8 and NKT+CD4, NKT+CD8, and NKT+DC lymphocytes were prepared. Assessment of the T helper cell type 1 (Th1)/Th2 cytokine secretion paradigm in vitro was performed before and following exposure to the antigen. Adoptive transfer of ex vivo immune-programmed lymphocytes from each group was performed into recipient mice, followed by colitis induction. Ex vivo exposure of NKT cells harvested from mice with colitis-to-colitis proteins [colitis-extracted proteins (CEP)] led to a Th2 cytokine shift. The interleukin (IL)-4/interferon-{gamma} (IFN-{gamma}) ratio increased for NKT harvested from colitis-harboring mice following exposure to CEP. Adoptive transfer of NKT lymphocytes harvested from colitis-harboring mice, which were ex vivo-educated, significantly alleviated experimental colitis in vivo. Intrahepatic NKT lymphocytes increased significantly in mice transplanted with NKT lymphocytes harvested from colitis-harboring donor mice, which were ex vivo-exposed to CEP, similar to mice transplanted with NKT lymphocytes harvested from tolerized donors. Exposure of NKT cells to the disease-target antigen induced a significant increase in the IL-4/IFN-{gamma} cytokine ratio. Adoptive transfer of a relatively small number of immune-programmed NKT cells induced a systemic Th1 to Th2-immune shift and alleviated immune-mediated colitis.

Key Words: NK1.1 T cells • oral tolerance • experimental colitis


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INTRODUCTION
 
T lymphocytes expressing natural killer (NK) cell markers (NKT cells) exist in low numbers in the peripheral blood and most other tissues but are abundant in the liver and bone marrow. They are characterized as CD4+ or CD4–CD8–and CD16– and express {alpha}ß T cell receptor (TCR)int. Upon in vivo and in vitro stimulations, they produce large amounts of interleukin (IL)-4 and interferon-{gamma} (IFN-{gamma}), exhibit enhanced cytolytic activity, and have a regulatory role in T helper cell (Th) differentiation [1 2 ].

Inflammatory bowel disease is a chronic, relapsing condition of unknown origin, which exhibits a variety of autoimmune features. Abnormal intestinal epithelial cell barrier function, excessive production of Th1 or Th2 cytokines, and unrestrained activation of CD4+ TCR{alpha}ß+ T cells contribute to the pathogenesis of these disorders [3 ]. Rectal administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) induces experimental colitis, characterized by severe transmural and granulomatous inflammation similar to Crohn’s disease in humans [4 5 ]. Stimulated cells in the inflamed mucosa produce increased amounts of IFN-{gamma}, IL-2, and IL-12 and reduced amounts of IL-4. Anti-inflammatory cytokines such as IL-10 and IL-4 alleviate the disease [6 7 ]. A defect in systemic or intestinal regulatory T cells is associated with the pathogenesis of the immune-mediated bowel inflammation.

Oral administration of an antigen has been shown to prevent or alleviate several immune-mediated disorders [8 9 ]. In some models, it was more potent than other methods of peripheral tolerance induction [10 11 12 13 14 15 ]. In the experimental colitis model, induction of low-dose oral tolerance inhibited the host colonic inflammatory response, thereby alleviating the clinical, macroscopic, and microscopic manifestations of colitis [16 17 ]. Tolerance induction in this model induced an immunological shift from a Th1-proinflammatory to a Th2-anti-inflammatory type of response. Moreover, adoptive transfer of tolerance by transplantation of splenocytes from tolerized donors to sublethally irradiated recipients further supports the presence of suppressor cells in this setting [17 ].

NKT lymphocytes were shown to play a regulatory role in immune-mediated disorders [18 ]. Others and we have recently shown that this subset of lymphocytes may have a role in peripheral tolerance induction. Induction of peripheral tolerance via oral administration of an antigen, or FK506 treatment, was associated with significant increases in intrahepatic NKT lymphocyte proportions and cytotoxicity functions [19 20 21 ]. Depletion of NKT lymphocytes prevented the Th1 to Th2-immune shift, hindering the ability to induce immune tolerance in experimental colitis [22 ]. NKT lymphocytes were also suggested to play an active role in immune modulation of experimental colitis. Adoptive transfer of tolerized NKT cells mediated the transfer of tolerance to recipient mice and prevented the induction of disease [23 ]. Recent studies have shown that this subset of T lymphocytes can function in pro- and anti-inflammatory directions [24 ].

The aim of the present study was to determine the possibility of ex vivo-immune programming of NKT cells via exposure to disease-target antigen and to evaluate the in vivo effect of adoptive transfer of immune-programmed cells. The results of this study show that NKT cells play a regulatory role in experimental colitis. Ex vivo exposure of NKT cells to the disease-target antigen induced a Th1 to a Th2 shift in the cytokine paradigm in vitro. Adoptive transfer of ex vivo immune-programmed NKT cells alleviated immune-mediated colitis in recipient mice in vivo. These results suggest that alleviation of the immune-mediated disorder can be achieved via ex vivo-immune programming of NKT cells, even when harvested from animals with colitis.


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MATERIALS AND METHODS
 
Animals
Normal, inbred C57/B6 female mice, aged 2–4 months, were obtained from Harlan (Indianapolis, IN) and were maintained in the Animal Core of the Hadassah-Hebrew University Medical School (Jerusalem, Israel). Mice were maintained on standard laboratory chow and kept in 12 h light/dark cycles. The Hebrew University-Hadassah Institutional Committee for the Care and Use of Laboratory Animals approved all animal experiments.

Induction of colitis
TNBS–colitis was induced by rectal instillation of TNBS, 1 mg/mouse, dissolved in 100 ml 50% ethanol as described [17 ].

Preparation and administration of oral antigen
Colons were removed from TNBS-induced colitis mice, cut into small strips, and mechanically homogenized. After filtration through a 40-µm nylon cell strainer, intact cells were spun down and removed. Proteins were quantified by using a protein assay kit (Biorad, Munich, Germany). Colitis-extracted proteins (CEP) were introduced into the experimental groups described below by using a feeding atraumatic needle every other day for 11 days (a total of five doses).

Lymphocyte isolation
Splenocytes were isolated from all animals in all experimental groups and were prepared as described before [22 ]. Spleens were crushed through a stainless mesh (size 60, Sigma Chemical Co., St Louis, MO). Cell suspension was placed in a 50-ml tube for 3 min and washed twice in cold phosphate-buffered saline (PBS; 1250 rpm for 10 min), and debris was removed. The cells were resuspended in PBS, the cell suspension was placed through a nylon mesh presoaked in PBS, and unbound cells were collected. Cells were washed twice in 45 ml PBS (1250 rpm at room temperature). For splenocyte isolation, 20 ml histopaque 1077 (Sigma Diagnostics, St. Louis, MO) was slowly placed underneath the cells suspended in 7 ml PBS in a 50-ml tube. The tube was centrifuged at 1640 rpm for 15 min at room temperature. Cells at the interface were collected, diluted in a 50-ml tube, and washed twice with ice-cold PBS (1250 rpm for 10 min). The viability by trypan blue staining was more than 95%.

In vitro preparation of lymphocytes
Splenocytes were prepared and separated into four subsets of lymphocytes, CD4+, CD8+, and NKT cells. Cell separation was done using magnetic cell sorting (MACS). Specific microbeads were used for each subset of lymphocytes: CD3, CD4, and CD8 microbeads and anti-NK beads (Miltenyi Biotec, Bergisch Gladbach, Germany). For NKT cells, anti-CD3 and anti-NK1.1 were used. Immediately following lymphocyte isolation, triplicates of 2–5 x 104 cells/500 µl PBS were put into Falcon 2052 tubes.

For adoptive transfer of cells, cells were resuspended in 10 µl fetal calf serum (FCS) with 1:20 fluorescein isothiocyanate–anti-mouse NK1.1 antibody (NKR-P1C, PharMingen, San Diego, CA) and were mixed every 10 min for 30 min. Cells were washed twice in 1% bovine serum albumin (BSA) and were kept in 4°C until reading. For the control group, only 5 µl 1% BSA was added. Analytical cell sorting was performed on 1 x 104 cells from each group with a fluorescence-activated cell sorter (FACS; FACSTAR plus, Becton Dickinson, Oxnard, CA). Only live cells were counted, and background fluorescence from nonantibody-treated lymphocytes was deducted from the levels obtained. Gates were set on forward-scatters, and side-scatters excluded dead cells and red blood cells. The data were analyzed with the Consort 30 two-color contour plot program (Becton Dickinson) or the CELLQuest program.

Separation of dendritic cells (DC)
Magnetic beads conjugated to monoclonal hamster anti-mouse CD11c (Miltenyi Biotec) were used for DC separation in accordance with the manufacturer’s instructions.

Experimental groups
Three clusters of mice, consisting of 30 animals each, were studied (Table 1 ): Control, naive animals (Group A), animals with colitis (Group B), and animals with colitis that were orally tolerized (Group C). Forty-two different lymphocyte subpopulations were prepared (Table 2 ): NKT cells harvested from animals from Groups A–C, which were exposed in vitro to BSA (A1, B1, C1) or to CEP (A2, B2, C2); CD4 cells harvested from animals from Groups A–C, which were exposed in vitro to BSA (A3, B3, C3) or to CEP (A4, B4, C4); CD8 cells harvested from animals from Groups A–C, which were exposed in vitro to BSA (A5, B5, C5) or to CEP (A6, B6, C6); splenocytes harvested from animals from Groups A–C, which were exposed in vitro to BSA (A7, B7, C7) or to CEP (A8, B8, C8); a mixture of NKT+CD4 cells harvested from animals from Groups A–C, which were exposed in vitro to BSA (A9, B9, C9) or to CEP (A10, B10, C10); a mixture of NKT+CD8 cells harvested from animals from Groups A–C, which were exposed in vitro to BSA (A11, B11, C11) or to CEP (A12, B12, C12); and a mixture of NKT+DC cells harvested from animals from Groups A–C, which were exposed in vitro to BSA (A13, B13, C13) or to CEP (A14, B14, C14).


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  		Table 1. Experimental and Control Groups


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  		Table 2. In Vitro Cytokine Secretion by Subsets of Lymphocytes Pulsed with CEP

Splenocyte cultures
Lymphocytes were harvested from mice 10 days after colitis induction. Triplicates of 104 lymphocytes were prepared for each of the 42 combinations from three animals in each group (A–C). Lymphocytes were cultured in 24-well tissue-culture plates and cultured for 72 h in the presence of CEP or BSA (50 µg/ml). Both sets collected supernatant fluids for cytokine measurement by enzyme-linked immunosorbent assay (ELISA). For each of the experimental groups, the aim was to test whether a subset of lymphocytes harvested from an animal with untreated colitis can be re-educated in vitro to secrete IL-4 via exposure to the disease-associated antigen (CEP).

Adoptive transfer of immune-regulated lymphocytes
To determine the in vivo effect of ex vivo-immune programming of lymphocytes and based on the data from the in vitro studies described above, adoptive-transfer studies using four of the 42 lymphocyte subsets were performed: B1, B2, C1, and C2 (Table 3 ). Donor mice in Groups B and C were killed 14 days after induction of colitis, and single suspensions of NKT lymphocytes derived from spleens were prepared as described [13 14 ]. Cell separation was performed using MACS. Specific anti-NK microbeads were used (Miltenyl Biotec). Cells were exposed to BSA or CEP (Table 3) as described above. Donor lymphocytes from these four groups were resuspended in PBS before transplantation.


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  		Table 3. Donor and Recipient Groups for Adoptive Transfer of Immune-Programmed Lymphocytes

Recipient mice
Four groups of recipient mice consisting of 10 animals each were studied: B1R, B2R, C1R, and C2R (Table 3) . Recipient mice were sublethally irradiated with 300 rad total body irradiation 24 h before intravenous injection of 1 x 106 donor cells. All mice were treated with TNBS enemas 24 h following cell transplantation. Clinical, macroscopic, and histological parameters for colitis were determined 14 days following colitis induction, as described below.

Evaluation of the effect of adoptive transfer of immune-regulated lymphocytes on experimental colitis
The effect of tolerance induction in donor mice and of adoptive transfer of immune-regulated lymphocytes in recipient mice was evaluated by monitoring the following parameters for colitis and immune response to the disease-associated antigens:

Clinical assessment of colitis
Diarrhea was followed daily throughout the study.

Macroscopic score of colitis
Colitis assessment was performed 14 days following colitis induction using standard parameters. Four macroscopic parameters were determined: degree of colonic ulcerations, intestinal and peritoneal adhesions, wall thickness, and degree of mucosal edema. Each parameter was graded on a scale from 0 (completely normal) to 4 (most severe) by two experienced, blinded examiners.

Microscopic score of colitis
For histological evaluation of inflammation, distal colonic tissue (last 10 cm) was removed and fixed in 10% formaldehyde. Five paraffin sections from each mouse were then stained with hematoxylin–eosin by the use of standard techniques. The degree of inflammation on microscopic cross-sections of the colon was graded semiquantitatively from 0 to 4. Grade 0: Normal with no signs of inflammation; Grade 1: very low level of leukocyte infiltration; Grade 2: low level of leukocyte infiltration; Grade 3: high level of infiltration with high vascular density and bowel-wall thickening; Grade 4: transmural infiltrates with loss of goblet cells, high vascular density, wall thickening, and disruption of normal bowel architecture. Two experienced, blinded examiners performed grading.

Liver and spleen lymphocyte isolation from recipient mice
Splenocytes and liver lymphocytes were isolated as described previously with the following modifications [16 ]. The inferior vena cava was cut above the diaphragm, and the liver was flushed with 5 ml cold PBS until pale. Connective tissue and gallbladder were removed, and livers were placed in a 10-ml dish in cold, sterile PBS. Livers and spleens were crushed through a stainless mesh (size 60, Sigma Chemical Co.). Cell suspension was placed in a 50-ml tube for 3 min and washed twice with cold PBS (1250 rpm for 10 min), and debris was removed. Cells were resuspended in PBS, cell suspension was placed through nylon mesh presoaked in PBS, and unbound cells were collected. Cells were washed twice in 45 ml PBS (1250 rpm at room temperature). For liver and spleen lymphocyte isolation, 20 ml histopaque 1077 (Sigma Diagnostics) was slowly placed underneath the cells suspended in 7 ml PBS in a 50-ml tube. The tube was centrifuged in 1640 rpm for 15 min at room temperature. Cells at the interface were collected, diluted in a 50-ml tube, and washed twice with ice-cold PBS (1250 rpm for 10 min). Approximately 1 x 106 cells/mouse liver was recovered. The viability by trypan blue staining was more than 95%. Splenocytes and liver-associated lymphocytes were isolated from all animals in all recipient groups.

Flow cytometry analysis for determination of CD4+, CD8+, and NK 1.1+ T lymphocyte subsets
To characterize the subset of lymphocytes isolated from the liver and spleen, Limulus amoebocyte lysate and splenocytes were tested after lymphocyte isolation using anti-NK1.1, anti-CD4, anti-CD8, and anti-CD16 antibodies (PharMingen).

Cytokine secretion
Blood was drawn and centrifuged at 14,000 rpm, and cytokine levels were measured in the serum 14 days after induction of colitis. A "sandwich" ELISA using Genzyme Diagnostics kits (Cambridge, MA) measured IFN-{gamma}, IL-4, and IL-10 serum levels in accordance with the manufacturer’s instructions.

T cell proliferation assays
Sample collection, preparation, and testing were conducted as described above. Splenocytes were isolated by Ficoll gradient separation, grown in triplicates of 105 cells in RPMI with 10% FCS, and stimulated in vitro using 0.001 µg/ml, 0.1 µg/ml, or 1 µg/ml CEP. Five to 7 days later, methyl-H3thymidine was added to CEP-pulsed T cells (1 µCi/ml, Amersham Pharmacia Biotech, Little Chalfont, UK). T cell cultures were harvested following 18 h. Controls were incubated in the presence of 2.5 µg/ml phytohemagglutinin (PHA) or medium alone. Data were given as mean stimulation indices of triplicates and ±SEM, calculated from the ratios of incorporated radioactivities of T cell cultures expressed as counts per minute in the presence or absence of antigen.

IFN-{gamma} enzyme-linked immunospot (ELISPOT) assay
IFN-{gamma} spot-forming cells were determined using the ELISPOT assay (Mabtech, Nacka, Sweden) as described with the following modifications [22 ]. In brief, 96-well filtration plates, coated with a high protein-binding hydrophobic polyvinylidene disulfide membrane, were used (Millipore Corp., Bedford, MA). Plates were coated with 1-D1K anti-IFN-{gamma}-coating antibody (15 mg/ml, Mabtech) for 24 h at 4°C. Spleen-derived peripheral blood mononuclear cells (PBMC) were isolated by Ficoll gradient separation from 20 ml blood samples collected in acid citrate dextrose tubes and processed within 1 h. PBMC were washed twice in RPMI 1640 with 10% FCS. Cells were cultured in 96-well plates (1x105 cells/well) with RPMI 1640 and 10% FCS. Three triplicates were prepared with CEP (50 µg/ml), PHA (2.5 µg/ml), or RPMI without antigen. Plates were incubated for 48 h at 37°C and 5% CO2. Following washing, dilute-biotinylated antibodies (7-B6-1-biotin, Mabtech) were added in filtered PBS with 0.5% FCS to 1 µg/ml in a total volume of 100 µl/well. Plates were incubated for 3 h at room temperature. Following washing, 100 ml streptavidin-alkaline phosphatase was added, and plates were incubated for 90 min at room temperature. Following washing, a substrate was added (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium, BioRad, Richmond, CA) for 30 min until dark, red, purple spots emerged. Two independent investigators equipped with a dissection microscope counted dark spots, reflecting IFN-{gamma}-secreting clones. Results are expressed as means of triplicate IFN-{gamma}-secreting cells per 105 PBMC after subtracting the mean spots from wells without CEP antigens.

Statistical analysis
Results were analyzed using the standard Student’s t-test.


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RESULTS
 
Evaluation of the effect of oral tolerance induction in the experimental colitis model
Clinical assessment of colitis
A marked decrease in diarrhea was observed in tolerized mice fed with CEP (Group C). In contrast, mice fed with BSA suffered severe diarrhea (Group B). A follow-up observation of mouse body weight found a statistically significant increase in body weights among tolerized mice as compared with nontolerized mice (12.8% and 11.6% vs. 3.7% for naive, tolerized, and nontolerized mice, respectively; P<0.005).

Macroscopic grading of colitis
Induction of oral tolerance by the feeding of mouse-extracted, colitis-derived proteins markedly alleviated the macroscopic grading of colitis. Scores for tested macroscopic parameters of colitis included degree of colonic ulceration, intestinal and peritoneal adhesions, wall thickness, and degree of mucosal edema. The total macroscopic score was 0.91 ± 0.24 in Group C-tolerized mice as compared with 3.22 ± 0.15 in nontolerized mice in Group B (P<0.005).

Grading of histological lesions
Histological evaluation of bowel tissue showed a marked reduction in inflammatory response and mucosal ulcerations in tolerized mice with histological scores of 1.1 ± 0.6. In these mice, the sections were almost normal, as only minimal lymphocytic infiltration was detected. In contrast, severe inflammatory reactions were observed in bowel specimens taken from nontolerized mice with histological scores of 3.17 ± 0.56 (P<0.005).

Evaluation of the effect of in vitro exposure of subpopulations of lymphocytes to disease-target antigens on IL-4, IL-12, and IFN-{gamma}
Supernatant fluids were collected from all sets of triplicates, and cytokine levels were measured for all 42 subsets of lymphocytes from all experimental and control groups. NKT cells were harvested from Groups A–C animals, which were exposed in vitro to BSA (A1, B1, C1) or CEP (A2, B2, C2). A significant increase in IFN-{gamma} secretions from NKT cells harvested from animals with colitis (B1) compared with increased IL-4 secretion from NKT lymphocytes harvested from tolerized mice (C1, Table 2 ). Exposure of colitis-harvested NKT cells to CEP in vitro led to a shift in the cytokine-secretion profile, with a significant increase in IL-4 and decrease in IFN-{gamma} secretion (B2). The IL-4/IFN-{gamma} ratio increased significantly from 0.13 to 5.65 for Groups B1 and B2, respectively (Fig. 1A ). This ratio is similar to the one observed in tolerized animals (Groups C1 and C2). These results suggest that in vitro exposure of NKT cells to the disease-target antigen can alter the cytokine profile of these cells. A similar effect was observed for CD4 cells, which when harvested from Groups B and C animals exposed in vitro to CEP (B4, C4), showed a significant increase in IL-4 secretion. The IL-4/IFN-{gamma} ratio increased significantly from 0.10 to 4.26 for Groups B3 and B4, respectively (Fig. 1B) , similar to those observed in tolerized animals (Groups C4).



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  		Figure 1. Effect of in vitro exposure of subpopulations of lymphocytes to disease-target antigens on IL-4/IFN-{gamma} ratio. Supernatant fluids were collected from all sets of triplicates, and cytokine levels were measured for all 42 subsets of lymphocytes from all experimental and control groups. (A) The IL-4/IFN-{gamma} ratio increased significantly in Group B2. This ratio is similar to the one observed in tolerized animals (Groups C1 and C2). (B) A similar effect was observed for CD4 cells. The IL-4/IFN-{gamma} ratio increased significantly in Group B4 mice, similar to those observed in tolerized animals (Group C4). (C) No effect was observed when CD8 or splenocytes were exposed in vitro to CEP. CD8 cells harvested from animals from Groups A–C, exposed in vitro to CEP (A6, B6, C6), did not show a shift in the cytokine profile. Similarly, splenocytes harvested from animals from Groups A–C, exposed in vitro to CEP (A8, B8, C8), also did not manifest a change in cytokine secretion. (D) A mixture of NKT+CD4 cells harvested from animals from Groups A–C, exposed in vitro to BSA (A9, B9, C9) or to CEP (A10, B10, C10), did not show a change in the cytokine paradigm. Similarly, a mixture of NKT+CD8 cells harvested from animals from Groups A–C, exposed in vitro to BSA (A11, B11, C11) or to CEP (A12, B12, C12), and a mixture of NKT+DC cells harvested from animals from Groups A–C, which were exposed in vitro to BSA (A13, B13, C13) or to CEP (A14, B14, C14), did not induce a shift in cytokine secretion.

In contrast, no effect was observed when CD8 or splenocytes were exposed in vitro to CEP. CD8 cells harvested from Groups A–C animals exposed in vitro to CEP (A6, B6, C6) did not show a shift in the cytokine profile. Similarly, splenocytes harvested from animals from Groups A–C and exposed in vitro to CEP (A8, B8, C8) also did not manifest a change in cytokine secretion. The IL-4/IFN-{gamma} ratio was 0.80 and 0.81 for splenocytes harvested from mice in Groups B7 and B8, respectively (Fig. 1C) .

As the intercellular effects may determine the immunological environment of NKT cells, several mixtures of lymphocytes were prepared. A mixture of NKT+CD4 cells harvested from Groups A–C animals and exposed in vitro to BSA (A9, B9, C9) or to CEP (A10, B10, C10) did not show a change in the cytokine paradigm. Similarly, a mixture of NKT+CD8 cells harvested from Groups A–C animals and exposed in vitro to BSA (A11, B11, C11) or to CEP (A12, B12, C12) and a mixture of NKT+DC cells harvested from animals from Groups A–C, which were exposed in vitro to BSA (A13, B13, C13) or to CEP (A14, B14, C14), also did not induce a shift in cytokine secretion (Fig. 1D) .

IL-12 levels increased in colitis-harboring mice (Groups B and C). No statistically significant differences were observed between different groups (Table 2) .

Evaluation of the effect of adoptive transfer of in vitro immune-programmed lymphocytes on experimental colitis
To determine the in vivo effect of ex vivo-immune programming of lymphocytes and based on the data from the in vitro studies described above, adoptive-transfer studies using four of the 42 lymphocytes subsets were performed: B1, B2, C1, C2 (Table 3) . Adoptive transfer of NKT cells exposed to CEP led to a significant alleviation of experimental colitis.

Clinical assessment of colitis
A marked decrease in diarrhea was observed in mice transplanted with NKT lymphocytes harvested from colitis-harboring donor mice, which were ex vivo-exposed to CEP (recipient Group B2R), similar to mice transplanted with NKT lymphocytes harvested from tolerized donors (recipient Groups C1R and C2R). In contrast, recipient mice transplanted with NKT cells from donors fed with BSA suffered severe diarrhea (Group B1R).

A follow-up of mice body weight found a statistically significant increase in body weights among mice transplanted with NKT lymphocytes harvested from colitis-harboring donor mice, which were ex vivo-exposed to CEP (recipient Group B2R), similar to mice that were transplanted with NKT lymphocytes harvested from tolerized donors (recipient Groups C1R and C2R, 11.3%, 12.1%, and 12.2%, for Groups B2R, C1R, and C2R, respectively). In contrast, recipient mice transplanted with NKT cells from donors fed with BSA showed only minimal increase in body weights (Group B1R, 3.5%; P<0.005).

Microscopic grading of colitis
Adoptive transfer of NKT cells that were ex vivo-exposed to CEP markedly alleviated the microscopic grading of colitis. Colitis microscopic score in mice transplanted with NKT lymphocytes harvested from colitis-harboring donor mice exposed ex vivo to CEP (recipient Group B2R) was low, similar to mice transplanted with NKT lymphocytes harvested from tolerized donors (recipient Groups C1R and C2R, 0.4±0.03, 1.08±0.09, and 1.43±0.11 for Groups B2R, C1R, and C2R, respectively). In contrast, recipient mice transplanted with NKT cells from donors fed with BSA showed a high histological score (Group B1R, 2.61±0.25; P<0.005; Figs. 2 and 3 ).



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  		Figure 2. Effect of adoptive transfer of in vitro immune-programmed lymphocytes on the microscopic score of experimental colitis. Adoptive transfer of NKT cells, which were ex vivo-exposed to CEP, markedly alleviated the macroscopic grading of colitis. The colitis microscopic score in mice transplanted with NKT lymphocytes harvested from colitis-harboring donor mice ex vivo-exposed to CEP (recipient Group B2R) was low, similar to that of mice transplanted with NKT lymphocytes harvested from tolerized donors.



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  		Figure 3. Effect of adoptive transfer of in vitro immune-programmed lymphocytes on experimental colitis. Mice transplanted with NKT lymphocytes harvested from colitis-harboring donor mice ex vivo-exposed to CEP (panel B2R) showed marked alleviation of colitis, similar to mice transplanted with NKT lymphocytes harvested from tolerized donors (panels C1R and C2R). In contrast, recipient mice transplanted with NKT cells from donors fed with BSA showed only severe colitis (panel B1R).

Serum cytokine levels
Mice in all recipient groups were tested for IFN-{gamma}, IL-4, and IL-10 serum levels 14 days following colitis induction. Adoptive transfer of NKT cells, which were exposed ex vivo to CEP, induced a significant decrease in IFN-{gamma} serum levels. IFN-{gamma} levels were 114.75 ± 15.8 in recipient mice transplanted with NKT cells from BSA fed donors and decreased significantly in mice transplanted with NKT lymphocytes harvested from colitis-harboring donor mice exposed ex vivo to CEP (recipient Group B2R), similar to levels in mice, which were transplanted with NKT lymphocytes harvested from tolerized donors (recipient Groups C1R and C2R, 41±4.24, 69±7.32, and 75±5.19 for Groups B2R, C1R, and C2R, respectively; P<0.005 in comparison with Group B1R; Fig. 4 ). No statistically significant differences were seen between the groups as for IL-4 and IL-10 serum levels [17±0.24, 5±0.32, 5±0.9, and 15±1.24; 32±3.24, 5±0.32, 26±2.19, and 33±2.64 for IL-4 and IL-10 for Groups B1R, B2R, C1R, C2R, respectively; P=not significant (NS); Fig. 4 ].



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  		Figure 4. Effect of adoptive transfer of in vitro immune-programmed lymphocytes on serum-cytokine levels. Adoptive transfer of NKT cells ex vivo-exposed to CEP induced a significant decrease in IFN-{gamma} serum levels. High IFN-{gamma} levels were found in recipient mice transplanted with NKT cells from BSA-fed donors.

T cell proliferation and ELISPOT assay
Splenocytes were harvested from recipient mice in all groups and exposed to CEP for proliferation and IFN-{gamma} ELISPOT assays. No significant differences were observed among the four recipient groups. CEP-specific T cell proliferation scores were 0.65 ± 0.04, 0.7 ± 0.02, 0.5 ± 0.1, and 1.03 ± 0.2 for Groups B1R, B2R, C1R, and C2R, respectively (P=NS; Fig. 5 ). The number of spot-forming colonies was zero for mice in Groups B1R, B2R, and C2R and two for mice in Group C1R (P=NS; Fig. 5 ).



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  		Figure 5. Effect of adoptive transfer of in vitro immune-programmed lymphocytes on T cell proliferation and ELISPOT assay. Splenocytes were harvested from recipient mice in all groups and exposed to CEP for proliferation and IFN-{gamma} ELISPOT assays. No significant differences were observed among the four recipient groups.

FACS analysis for subsets of T cells in the spleen and liver
FACS analysis for subsets of T lymphocytes was performed on splenocytes and liver-associated lymphocytes in mice in all recipient groups. For splenocytes, no statistically significant differences were observed among the four groups with regard to CD4 and CD8 proportions (20%, 22%, 16%, and 18% for CD4 and 12%, 20%, 11.89%, and 24.7% for CD8 for Groups B1R, B2R, C1R, and C2R, respectively; P=NS; Fig. 6 ). A trend toward an increase in NKT cells was observed (12%, 11%, and 12%, for Groups B2R, C1R, and C2R, respectively, as compared with 10% in Group B1R mice; P=NS; Fig. 6 ). Intrahepatic NKT lymphocytes increased significantly in Group B2R mice transplanted with NKT lymphocytes harvested from colitis-harboring donor mice, which were ex vivo-exposed to CEP, and in mice from Group C1R transplanted with NKT lymphocytes harvested from tolerized donors (3% and 0.79%, respectively) as compared with 0.4% in Group B1R mice transplanted with NKT cells from donors fed with BSA (P<0.005; Fig. 7 ).



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  		Figure 6. Effect of adoptive transfer of in vitro immune-programmed lymphocytes on FACS analysis for subsets of T cells in the spleen. FACS analysis for subset of T lymphocytes was performed on splenocytes and liver-associated lymphocytes in all recipient group mice. For splenocytes, no statistically significant differences were observed among the four groups with regard to CD4 and CD8 proportions.



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  		Figure 7. Effect of adoptive transfer of in vitro immune-programmed lymphocytes on FACS analysis for NKT cells in the liver. Intrahepatic NKT lymphocytes increased significantly in Group B2R mice transplanted with NKT lymphocytes harvested from colitis-harboring donor mice, which were ex vivo-exposed to CEP, and in mice from Group C1R transplanted with NKT lymphocytes harvested from tolerized donors as compared with 0.4% in Group B1R mice transplanted with NKT cells from BSA-fed donors.


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DISCUSSION
 
The results of the present study suggest that NKT cells have a regulatory role in immune-mediated colitis. Ex vivo exposure of NKT cells harvested from mice with colitis, to disease-target antigen, led to a shift in the cytokine paradigm, resulting in a significant increase in the IL-4/IFN-{gamma} cytokine ratio. Adoptive transfer of ex vivo-modulated NKT lymphocytes significantly alleviated colitis. These results suggest that NKT lymphocytes may have a significant effect on the peripheral immune response subject to the environment to which they are exposed.

NKT lymphocytes are not stably polarized. Multiple signaling pathways were identified for their activation. Upon different triggers, TCR rearrangement triggers Th1 and Th2 cytokine secretion from NKT cells [25 26 27 ]. NK1.1R or IL-12R may selectively promote Th1-type cytokine secretion [28 ]. In contrast, NKT cells can rapidly produce IL-4 and play a regulatory role in autoimmune response in experimental allergic encephalomyelitis and in nonobese diabetic mice models [29 30 ]. They are also involved in antigen presentation and in CD4+ T cell differentiation via secretion of a large amount of IL-4 upon in vivo stimulation [25 ]. It was recently shown that this subset of lymphocytes has a dual role in immune regulation and in switching the immune response in immunogenic or tolerogenic directions [22 ]. It was suggested that the environment in which NKT cells are activated may determine their function. In a tolerized environment, NKT cells support immune hyporesponsiveness, and in a nontolerized environment, they support a proinflammatory, immune response [31 ]. In the experimental colitis model, NKT response was dependent on presentation of the disease-target antigen in the bowel. In the presence of antigen, this subset of lymphocytes supported disease alleviation. Their depletion induced disease exacerbation with a decrease in the CD4+IL-4+/CD4+IFN-{gamma}+ T lymphocyte ratio. In contrast, in the absence of antigen in the bowel, NKT lymphocytes played a role in induction of inflammation. Their depletion ameliorated colitis and increased the CD4+IL-4+/CD4+IFN-{gamma}+ T lymphocyte ratio [31 ]. Thus, the environment in which NKT cells are being activated, the types of stimulations, or signaling receptors may determine their function.

As a regulatory subtype of lymphocytes that can function in different ways depending on the environment to which they are exposed, we hypothesized that NKT cells can be ex vivo "immune-educated." The results of the first part of the present study showed that exposure of NKT cells harvested from mice with immune-mediated colitis to CEP in vitro led to a shift in cytokine secretion profile, with a significant increase in IL-4 and decrease in IFN-{gamma} secretion (Group B2). Following exposure to antigen, the IL-4/IFN-{gamma} ratio significantly increased in NKT harvested from colitis-harboring mice from Groups B1 and B2 and was similar to the one observed in tolerized animals (Groups C1 and C2). These results suggest that in vitro exposure of NKT cells to the disease-target antigen can alter the cytokine profile of these cells. A similar effect was observed for CD4 cells, which when harvested from Groups B and C animals exposed in vitro to CEP (B4, C4), also showed an increase in IL-4 secretion. The IL-4/IFN-{gamma} ratio increased for Groups B3 and B4, similar to those observed in tolerized animals (Group C4). However, these data did not take into account the heterogenicity of NKT lymphocyte subsets [25]. As these cells can be CD4+ or double-negative, the data may not reflect actual function of a different subset of regulatory cells. As differentiation between subtypes of CD4 lymphocytes was not performed, other groups of regulatory cells cannot be ruled out. In contrast, no effect was observed when CD8 or splenocytes were exposed in vitro to CEP.

Based on the in vitro data, we determined whether this alteration could induce a systemic immune effect in vivo. The results of the second part of the study showed that adoptive transfer of NKT lymphocytes, which were ex vivo-educated, significantly alleviated experimental colitis in vivo. Recipient mice transplanted with NKT cells harvested from colitis-harboring mice and exposed ex vivo to CEP manifested weight gain, significant decrease in the macroscopic and microscopic scores for colitis, and decrease of IFN-{gamma} serum levels (Group B2R). Intrahepatic NKT cells significantly increased in these mice with a slight increase in the periphery, suggesting a role for intrahepatic NKT cells in the tolerizing process. Similar results were observed in recipients of NKT cells harvested from tolerized donors exposed to CEP (Group C1R). Adoptive transfer of NKT cells harvested from tolerized donors exposed to BSA somewhat attenuated the Th1-to-Th2 shift manifested by the clinical, histological, and immunological effects (Group C2R). These data suggest that NKT cells are a unique subset of regulatory T cells and that their cytokine secretion pattern can be altered ex vivo. In addition, once this immune phenotypic alteration has occurred, a relatively small number of NKT cells can induce a systemic Th1 to Th2-immune shift in vivo.

The cross-talk between different types of immune cells may be one of the factors that determines the "immune environment" for NKT cells. To determine whether other effector cells have a contributory or disturbing role in the process, several mixtures of lymphocytes were prepared. Mixtures of NKT+CD4, NKT+CD8, or NKT+DC cells, which were exposed in vitro to CEP, did not induce a change in the cytokine secretion pattern by NKT cells. CD4, CD8, and DC inhibited the cytokine secretion shift induced by CEP on NKT lymphocytes.

It is possible that different stimuli determine the type of cytokine secretion and/or cytotoxicity function of NKT lymphocytes [32 ]. The liver is known to play a role in systemic immune regulation [33 34 35 ]. It was recently proposed that the liver is a major site of T cell destruction and that the failure of this process with leakage of T cells from the liver to peripheral lymphoid tissue may exacerbate autoimmunity in the lpr/lpr mouse model [34 35 36 37 ]. As NKT cells are abundant in the liver, the results of the present study suggest that the intrahepatic environment may determine the function of NKT cells [33 ]. This subtype of lymphocytes may mediate the peripheral tolerizing effect of the liver in immune-mediated disorders. NKT cells may be involved in maintaining a balance between anti-inflammatory and proinflammatory lymphocytes via cytokine secretion or via the killing of pro- or anti-inflammatory subsets of lymphocytes. They may also be involved in the determination of antigen presentation or Th cell differentiation. It is possible that in a tolerized environment, NKT cells are involved in killing sensitized, proinflammatory cells in addition to their IL-4-mediated, anti-inflammatory cytokine secretion [37 ]. In a nontolerized environment, they are involved in killing anti-inflammatory cells in addition to IFN-{gamma} secretion.

NKT cells are innate lymphocytes, which are specific for glycolipid antigens bound by the major histocompatibility complex class I-like protein CD1d. One striking property of NKT cells is their capacity to modulate adaptive-immune responses [18 ]. NKT lymphocytes mediate a rapid reaction to the glycolipid drug {alpha}-galactosylceramide ({alpha}GalCer), which triggers the release of large amounts of cytokines into the serum within hours, starting with IL-4. NKT cells were shown to play an important role in protection against malignancy, infections, allograft rejection, and other immune-mediated disorders [1 18 ]. Recent preclinical studies have revealed significant efficacy of NKT cell ligands such as the glycolipid {alpha}GalCer for treatment of metastatic cancers and infections and for prevention of autoimmune diseases [38 39 40 41 42 43 44 45 46 ]. In some of these, a defect in regulatory subsets of cells, such as NKT cells, may be an important factor in disease pathogenesis. Appropriate stimulation of NKT cells could be exploited for prevention or treatment of human diseases. The results of the present study suggest that NKT cells are a regulatory subset of lymphocytes that can influence the Th1/Th2 profile of immune responses via IFN-{gamma} proinflammatory or via IL-4 anti-inflammatory cytokine secretion. Modulation of the immune environment ex vivo, via exposure to an antigen, or isolation of NKT cells from other subsets of "disturbing" effector cells may serve as a possible means of altering the malfunctioning of regulatory T cells in immune-mediated disorders.

In conclusion, NKT cells are actively involved in distinct, immune-regulatory mechanisms and may modulate the type of effector cells and the Th1/Th2 paradigm in immune-mediated disorders. The data of the present study suggest that regulation of an immune-mediated colitis can be achieved via ex vivo-immune education of NKT cells. This process may involve cytokine secretion or other measures of cell signaling. Further studies are required to determine if the procedure described here may serve as a mode of treatment for disorders in which peripheral immune balance is defective.


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ACKNOWLEDGEMENTS
 
This work was supported in part by the following grants: the Israel Science Foundation, the Roaman-Epstein Liver Research Foundation, and ENZO Biochem Inc. (to Y. I.).


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FOOTNOTES
 
1 These authors contributed equally to this manuscript. Back

Received July 28, 2003; revised September 2, 2003; accepted September 4, 2003.


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