The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Namba, K.
Right arrow Articles by Onoé, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Namba, K.
Right arrow Articles by Onoé, K.
The Journal of Immunology, 2000, 165: 2962-2969.
Copyright © 00 by The American Association of Immunologists

Amelioration of Experimental Autoimmune Uveoretinitis by Pretreatment with a Pathogenic Peptide in Liposome and Anti-CD40 Ligand Monoclonal Antibody1

Kenichi Namba*,{dagger}, Kazumasa Ogasawara2,*, Nobuyoshi Kitaichi*,{dagger}, Taiki Morohashi*, Yoichi Sasamoto{dagger}, Satoshi Kotake{dagger}, Hidehiko Matsuda{dagger}, Kazuya Iwabuchi*, Chikako Iwabuchi*, Shigeaki Ohno{ddagger} and Kazunori Onoé3,*

* Division of Immunobiology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan; {dagger} Department of Ophthalmology, School of Medicine, Hokkaido University, Sapporo, Japan; and {ddagger} Department of Ophthalmology, Yokohama City University School of Medicine, Yokohama, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have defined a peptide K2 (ADKDVVVLTSSRTGGV) that corresponds to residues 201–216 of bovine interphotoreceptor retinoid-binding protein and induces experimental autoimmune uveoretinitis (EAU)4 in H-2Ak-carrying mice (H-2Ak mice). In this study, we attempted to ameliorate EAU in the H-2Ak mice without nonspecific suppression of T cell responses. Preceding s.c. administration of liposomes including K2 (liposomal K2) specifically inhibited subsequent generation of T cell response to K2. The same result was obtained with a combination of OVA323–339 peptide and the OVA-specific TCR-transgenic T cells. It was suggested that the inhibition was mainly attributed to peripheral anergy induction of T cells specific for the peptide Ag, although specific cell death might also be involved in the inhibition. Pretreatment with liposomal K2 also considerably abolished IFN-{gamma} production but not IL-4 production. The specific inhibitory effect of the pretreatment with liposomal peptide was augmented by a simultaneous administration of anti-CD40 ligand (anti-CD40L) mAb. Moreover, it was shown that the pretreatment with liposomal K2 reduced both the incidence and severity of the subsequent K2-induced EAU, and the simultaneous administration of anti-CD40L mAb augmented this preventive effect by liposomal K2. Our findings demonstrate that the s.c. administration of liposomal pathogenic peptide and anti-CD40L mAb can be applied to preventing autoimmune diseases without detrimental nonspecific suppression of T cell responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Experimental autoimmune uveoretinitis (EAU)4 is an organ-specific, T cell-mediated autoimmune disease that can be induced in several animal models by immunization with retinal Ag, e.g., interphotoreceptor retinoid-binding protein (IRBP) emulsified in CFA, or by the adoptive transfer of retinal Ag-specific T lymphocytes into naive syngeneic recipients (1, 2, 3). Histopathology in the animal model resembles several human endogenous uveoretinitis (e.g., Behçet’s disease, sarcoidosis, Vogt-Koyanagi-Harada syndrome, birdshot retinochoroidopathy, and sympathetic ophthalmia) and is characterized by posterior retinal and choroidal lesions, photoreceptor damage, vasculitis, vitritis, granuloma formation, and varying degrees of anterior chamber infiltration (4, 5, 6). However, cumbersome IRBP preparation from bovine eyes and complex antigenicity of the IRBP had been an obstacle to precise immunological analyses of EAU. To circumvent this obstacle, referring to the H-2Ak-binding motif, we have defined a peptide, K2, in bovine IRBP that induces EAU in H-2Ak mice (7).

A desirable therapy of autoimmune diseases involving autoreactive CD4+ T cells would be selective inactivation, elimination, or functional deviation of the disease-causing T cells. Various approaches have been used to regulate Ag-specific responses in experimental allergic encephalomyelitis (EAE), which is another type of organ-specific autoimmune animal model. It was reported that administration of a low number of encephalitogenic T cells (8), immunization with peptide derived from TCR of encephalitogenic clones (9), or administration of mAb specific for TCR of encephalitogenic clones (10) prevented development of EAE. It was also reported that an i.p. injection or repeated s.c. injections of an encephalitogenic peptide in IFA ameliorated EAE by inducing anergy of encephalitogenic T cells (11, 12). In contrast, Forsthuber et al. (13) demonstrated that the administration of encephalitogenic peptides in IFA induced differentiation of potentially encephalitogenic T cells into nonencephalitogenic, possibly protective cells of Th2 phenotype. The precise mechanism underlying the induction of T cell tolerance by administration of the peptide in IFA is still unclear.

In this study, we attempted to prevent K2-induced EAU by the preceding administration of liposomal K2. We used liposome (phosphatidylcholine/phosphatidylserine = 7:3) instead of IFA because we observed that the liposome promoted effective incorporation of peptide Ag into dendritic cells (DC), a professional APC (14, 15). We report herein that the liposomal K2 induces T cell tolerance more efficiently than K2 in IFA and inhibits EAU induction. Furthermore, we show that simultaneous administration of liposomal K2 and anti-CD40 ligand (CD40L) mAb inhibits considerably the development of EAU as compared with the administration of liposomal K2 alone.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

B10.BR female mice (H-2Ak mice) and BALB/c mice (H-2Ad mice) were obtained from Shizuoka Laboratory Animal Corporation (Hamamatsu, Japan). OVA-specific TCR-transgenic mice (DO11.10 TCR mice) (16) were obtained from Dr. Dennis Loh (Washington University, St. Louis, MO) and were maintained in a specific pathogen-free condition of our animal facility at Hokkaido University.

Peptides and adjuvants

Peptide K2 (ADKDVVVLTSSRTGGV) that corresponds to bovine IRBP201–216, and OVA323–339 peptide (ISQAVHAAHAEINEAGR) were synthesized using automatic peptide synthesizer PSSM-8 (Shimadzu, Kyoto, Japan) and purified by reverse-phase HPLC (Waters Japan, Tokyo, Japan) as previously described (7). CFA, IFA, and Mycobacterium tuberculosis H37Ra were purchased from Difco (Detroit, MI). Bordetella pertussis inactive bacterial suspension was purchased from Wako Pure Chemical Industries (Tokyo, Japan).

Liposome and pretreatment

Multilamellar liposome was prepared as previously described (17). Because preliminary experiments showed that 50- to 200-nmol K2 peptides exerted almost the same levels of influence, 100-nmol peptides were used throughout the study. The peptide (100 nmol) was mixed with 1.25 µmol of phosphatidylserine (Avanti Polar Lipids, Alabaster, AL) and 2.75 µmol of phosphatidylcholine (Avanti Polar Lipids) in chloroform. In a glass tube, the mixture was blown by N2 gas, evaporated at 40°C, and aspirated for 3 h. The peptide incorporated in multilamellar liposome (liposomal peptide) that adhered to the inside of the glass tube was dissolved in 300 µl PBS by vortexing and sonication with or without anti-CD40L mAb (50 µg; PharMingen, San Diego, CA). The dose (50 µg) of anti-CD40L mAb was determined by preliminary experiments in which 50–500 µg/mouse mAb showed the same level of inhibition. The liposomal peptide with or without anti-CD40L mAb, liposome alone, or PBS (as a negative control) was s.c. injected into the backs of mice (three mice per group) (pretreatment) 10 days before immunization as described below. One group of mice was treated with peptide (100 nmol) in IFA.

Immunization

To analyze T cell proliferative response, B10.BR mice were immunized in the footpads and the tail base with peptide (50 nmol) in emulsion with CFA (1:1, v/v) (7). To induce EAU, B10.BR mice were immunized in the footpads and the tail base with 100 nmol of peptide K2 in CFA with the addition of 2.5 mg/ml of M, tuberculosis H37Ra. Concurrently, B. pertussis inactive bacterial suspension (1010 bacilli) in a volume of 100 µl was injected i.p. as an additional adjuvant (7).

T cell-proliferative responses

Ten days after immunization, T cell-enriched fractions from the draining lymph nodes pooled from three mice in each group were prepared by passing the dispersed cells over nylon wool columns. These cells (4 x 105/well) were cultured with 30 Gy-irradiated syngeneic spleen cells as APC and various concentrations of peptide or 5 µg/ml of purified protein derivative of tuberculin (PPD; Japan BCG Prod. Co., Tokyo, Japan) in a 96-well flat-bottom microtiter plate for 72 h. T cell proliferation was determined by [3H]thymidine incorporation in triplicate as previously described (7), and the data were presented as the mean incorporation ±SD with the background value (medium alone) subtracted ({Delta}cpm). Relative response (RR) was calculated in the following formula: RR = {Delta}cpm of the responses to K2/{Delta}cpm of the responses to PPD.

Cell transfer and immunization

BALB/c mice were i.v. injected with DO11.10 spleen cells (5 x 106/mouse) that had been depleted of CD8+ T cells by anti-CD8 mAb plus complement (14), and were designated as (DO11.10->BALB/c) mice. After 3, 6, and 9 days, liposomal OVA323–339 (50 nmol), liposome alone, or OVA323–339 in IFA was s.c. administered to the (DO11.10->BALB/c) mice. Three days after the last pretreatment, these mice were immunized with OVA323–339 (20 nmol) in CFA. Three days later, the OVA323–339-specific T cell responses and cytokine productions were analyzed. In a particular experiment, a single cell suspension of the draining lymph nodes of (DO11.10->BALB/c) mice was prepared and analyzed for expressions of CD4 and KJ1-26, a clonotypic mAb for DO11.10 TCR. The number of KJ1-26+CD4+ T cells in the cell suspension was calculated as described elsewhere (18). The responsiveness of the T cells to OVA323–339 was expressed as {Delta}cpm per 5000 CD4+ DO11.10 (KJ1-26+) T cells.

Cytokine ELISA

T cells from B10.BR mice (three mice per group) that had been pretreated with liposomal K2 with or without anti-CD40L mAb and then immunized with K2 in CFA were cultured with APC in the presence of K2 (30 µM) for 3 days. Amounts of IFN-{gamma}, IL-2, and IL-4 in the culture supernatants (CS) were quantitated with a Cytoscreen Immunoassay kit (BioSource International, Camarillo, CA) for mouse IFN-{gamma}, IL-2, and IL-4, respectively, as previously described (14, 19). Amounts of cytokines in CS of (DO11.10->BALB/c) lymph node cells were also quantitated as described above.

Clinical and histological assessment of EAU

From 10 days after immunization, funduscopic examinations of mice were performed every other day. Tropicamide and neo-synephrine saline (0.5%) were applied to eyes of mice. Five minutes later, after confirming mydriasis of the eye, fundus of the eye was examined with a Bonnoscope and Super Field NC Lens (Volk Optical, Mentor, OH). The clinical assessments were performed by two ophthalmologists in a blind fashion. Vessel dilatation, vessel white focal lesions, vessel white linear lesions, retinal hemorrhages, and retinal detachment were determined. According to the severity of these findings, EAU clinical scores were graded 0–4 as described by Thurau et al. (20) (Table IGo).


View this table:
[in this window]
[in a new window]
  		Table I. Clinical score of EAU in the mouse

 
At day 37 after immunization, eyes were enucleated and fixed for 1 h in 4% phosphate-buffered glutaraldehyde and transferred into 10% phosphate-buffered formaldehyde until processing. Fixed tissues were processed and the histological severity was graded on a scale of 0–4 as reported previously (7).

Statistical analysis

The data of mean thymidine uptake (cpm) or mean EAU clinical score were analyzed with Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pretreatment with liposomal K2 peptide decreased K2-specific T cell responses

B10.BR (H-2Ak) mice were s.c. administered PBS, liposome, K2 emulsified with IFA, or liposomal K2, and, 10 days later, immunized in the footpads with K2 emulsified with CFA. A representative result from three separate experiments is shown in Fig. 1Go. In the PBS- and liposome-pretreated mice, K2-primed T cells reached almost the same levels of response against K2 (Fig, 1, a and b). As reported earlier by Gaur et al. (11), the pretreatment with K2 in IFA considerably decreased the K2-specific T cell responses (Fig. 1Goc). It should be noted that the pretreatment with liposomal K2 reduced T cell responses more profoundly than that with K2 in IFA (Fig. 1God). Almost the same levels of PPD-specific T cell responses were detected in these mice pretreated with PBS, liposome alone, K2 in IFA, or liposomal K2 (5689, 5917, 7567, or 6619 {Delta}cpm, respectively). These results indicated that the inhibition of T cell responses by the pretreatment with s.c. administration of K2 in IFA or liposomal K2 was specific for the peptide used in the pretreatment. To evaluate the K2-specific inhibition, we divided the values of T cell responses to K2 by those to PPD. These ratios, termed RR, were shown in Fig. 1Goe (see Materials and Methods). It seems that liposomal K2 diminishes the K2-specific T cell responses more profoundly than K2 in IFA.



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 1. Inhibition of the proliferative responses of K2-primed T cells by pretreatment with liposomal K2. B10.BR mice (three mice per group) were pretreated with PBS (a), liposome alone (b), K2 peptide in IFA (c), or liposomal K2 (d). Ten days later, the pretreated mice were immunized with K2 in CFA. The proliferative T cell responses were quantitated as described in Materials and Methods. To evaluate the K2-specific inhibition by the pretreatment, the values of the T cell response to K2 were divided by those to PPD (e).

 
Time course of the inhibitory effect of pretreatment with liposomal K2 on K2-specific T cell responses

We then examined the time course of the inhibitory effect of the liposomal K2 pretreatment on the K2-specific T cell responses. Fig. 2Go shows a representative result obtained with two separate experiments. This figure illustrates T cell-proliferative responses in mice pretreated with liposomal K2 at different times. When H-2Ak mice were immunized with K2 emulsified in CFA 3 days after the pretreatment with liposomal K2 (-3), the T cells hardly responded to K2. The Ag-specific inhibitory effect of the liposomal K2 continued for 2–3 wk, although the inhibitory effect of the liposomal K2 was reduced compared with that seen in the 3-day group (Fig. 2Go). The inhibition of the K2-specific T cell responses was not seen at all when the s.c. administration of liposomal K2 was conducted 3 days after the immunization with K2 in CFA (+3). These findings demonstrate that the substantial inhibitory effect of the pretreatment with liposomal K2 on the subsequent K2-specific T cell responses lasted 2–3 wk.



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 2. Time course of inhibition of K2-specific T cell responses by the pretreatment with liposomal K2. B10.BR mice (three mice per group) were treated with liposomal K2. After 3, 14, or 21 days, the pretreated mice were immunized with K2 in CFA, and, 10 days later, the primed T cells were stimulated with K2 in vitro. In one group, mice were pretreated 3 days after immunization with K2 in CFA. C, Pretreatment with liposome alone; +3, Administration of liposomal K2 3 days after immunization; -3, -14, or -21, Pretreatment 3, 14, or 21 days before immunization, respectively.

 
Elucidation of mechanism underlying the specific T cell inhibition

To elucidate the mechanism underlying the specific inhibition of T cell responses by the pretreatment with liposomal peptide, we analyzed the inhibitory effect using another peptide Ag and TCR-transgenic T cells. In this experiment, a combination of a peptide, OVA323–339, which can be recognized by T cells bearing the transgenic DO11.10 TCR in the context of H-2Ad (16), and BALB/c (H-2Ad) mice transferred with the DO11.10 T cells, was used. It has been demonstrated that BALB/c mice-transferred DO11.10 T cells (DO11.10->BALB/c) are useful for analyzing the mechanism underlying T cell tolerance in vivo (21, 22, 23). In these mice, the T cells expressing DO11.10 TCR show normal lymphocyte functions and are readily detected with a clonotypic mAb, KJ1-26.

(DO11.10->BALB/c) mice were pretreated with liposomal OVA323–339, liposome alone, or OVA323–339 emulsified with IFA three times. Three days after the last pretreatment, these mice were immunized with OVA323–339 in CFA, and, 3 days later, the OVA323–339-specific T cell responses were analyzed. At this stage, normal BALB/c mice that had not received DO11.10 T cells generated no T cell responses to OVA323–339 (data not shown). Fig. 3Goa shows results of two representative mice in each group. Pretreatment with liposomal OVA323–339 considerably inhibited the proliferative responses of the lymph node cells. The inhibition was again more profound than that seen in mice pretreated with peptide in IFA (Fig. 3Goa). When amounts of IL-2 in the CS were quantitated, almost complete inhibitions were observed in (DO11.10->BALB/c) mice pretreated with liposomal peptide, and moderate inhibitions were seen in mice treated with peptide in IFA (Fig. 3Gob). Similar results were obtained with IFN-{gamma} production (data not shown).



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 3. Inhibition of OVA-specific T cell responses by liposomal peptide in mice adoptively transferred OVA-specific TCR-transgenic T cells. BALB/c mice that had been i.v. injected with KJ1-26-positive T cells (5 x 106/mouse) from DO11.10 TCR-transgenic mice were s.c. administered either liposomal OVA323–339 (P1, P2; the numbers indicate individual mice), liposome alone (C1, C2), or OVA323–339 in IFA (IFA1, IFA2) 3, 6, and 9 days after the transfer. Three days after the last treatment, these mice were immunized with OVA323–339 in CFA. Three days later, T cell proliferation to OVA323–339 was analyzed (a). Amounts of IL-2 in the CS were analyzed (b). Liposomal peptide-treated (P1, P2) or liposome alone-treated (C) mice were immunized with OVA323–339 in CFA, and 3 days later KJ1-26+CD4+ T cells in the draining lymph nodes were quantitated by FACS analysis, and the proliferative responses against OVA323–339 were examined (c). The mean cpm values at each peptide dose were adjusted to those per 5000 KJ1-26+CD4+ T cells in the culture. (*, p < 0.05 vs C)

 
Then, to evaluate the responsiveness per T cell, (DO11.10->BALB/c) mice that had been pretreated with liposomal OVA323–339 or liposome alone were immunized with OVA323–339 in CFA, and both the proportion of KJ1-26+CD4+ cells and the proliferative responses were quantitated. The proportion of KJ1-26+CD4+ T cells recovered from the lymph nodes of liposomal peptide-pretreated mice was lower than that in liposome alone-pretreated mice (data not shown). Then {Delta}cpm values were adjusted to those per 5000 KJ1-26+CD4+ T cells. Fig. 3Goc shows a representative result from two separate experiments. DO11.10 T cells from liposomal OVA323–339-pretreated (DO11.10->BALB/c) mice (P1 and P2) showed significantly low responsiveness as compared with that in the liposome alone-pretreated (DO11.10->BALB/c) mice (C). Almost the same result was obtained with the other experiment (data not shown). These findings suggest that the s.c. administration of liposomal OVA323–339 induces anergy and/or perhaps the depletion of KJI-26+CD4+ T cells.

Additional effect of anti-CD40L mAb administration on inhibition of specific T cell responses by pretreatment with liposomal K2

B10.BR mice (three mice per group) were pretreated with liposome alone, liposomal K2, or liposomal K2 plus anti-CD40L mAb and, 2 or 3 wk later, were immunized with K2 in CFA. After 10 days, T cell-proliferative responses were analyzed. Fig. 4Goa shows a representative result from two separate experiments. Again, liposomal K2 induced considerable inhibition of T cell responses to K2. The inhibitory effect of the liposomal K2 on the induction of K2-specific T cell responses was slightly but significantly augmented by the addition of anti-CD40L mAb to the liposomal K2 (Fig. 4Goa). Fig. 4Gob shows a result of another set of experiments. Considerable inhibitory effects of the liposomal K2 and anti-CD40L mAb on the K2-specific T cell responses were seen even 3 wk after the pretreatment, although the degree of inhibition appeared to be decreased as compared with that in mice immunized 2 wk after the pretreatment. Thus, simultaneous administration of anti-CD40L mAb resulted not only in the augmented inhibition of K2-specific T cell response but also in the prolongation of the inhibitory effect of the liposomal K2. Treatment with anti-CD40L mAb alone 3 wk before immunization with K2 in CFA showed no inhibition on the induction of K2-specific T cell responses (Fig. 4Goc).



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 4. Augmented K2-specific inhibition by simultaneous administration of anti-CD40L mAb and liposomal K2. a, B10.BR mice were pretreated with liposomal K2 (P), liposomal K2 and anti-CD40L mAb (PA), or liposome alone (C). Two weeks after the pretreatment, the B10.BR mice were immunized with K2 in CFA, and, 10 days later, T cell proliferation was evaluated (*, p < 0.01 vs P; **, p < 0.05 vs P). b, B10.BR mice were pretreated with liposomal K2 and anti-CD40L mAb. These mice were immunized with K2 in CFA 14 or 21 days later, and, after 10 days, the [3H]thymidine uptake was evaluated. C, Pretreatment with liposome alone; -14 or -21, pretreatment 14 or 21 days before immunization (*, p < 0.01 vs -21; **, p < 0.05 vs -21). c, B10.BR mice were pretreated with liposomal K2 and anti-CD40L mAb (PA), anti-CD40L mAb alone (A), or PBS (C). Three weeks after the pretreatment, the B10.BR mice were immunized with K2 in CFA, and the T cell proliferative response was determined. (*, p < 0.01 vs C)

 
Inhibition of Th1-type cytokine production by pretreatment with liposomal K2

We examined whether inhibition of K2-specific T cell-proliferative responses was associated with the inhibition of cytokine production. K2-primed T cells from B10.BR mice that had been pretreated as described above were cocultured with APC and 30 µM K2 peptide, and amounts of IFN-{gamma}, IL-2, and IL-4 in the CS were determined. Fig. 5Goa shows that T cells from mice pretreated with liposomal K2 in vivo produced a significantly smaller amount of IFN-{gamma} than those treated with PBS or liposome alone (controls). In contrast, no influence on the level of IL-4 production in the CS was observed in these mice (Fig. 5Gob). Thus, the reduction of IFN-{gamma} but not of IL-4 appeared to alter the IFN-{gamma}-IL-4 ratio and polarize toward a Th2 shift.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 5. Depressed production of IFN-{gamma} but not of IL-4 in CS of T cells from liposomal K2-pretreated mice. B10.BR mice (three mice per group) were pretreated with liposomal K2 plus anti-CD40L mAb, liposomal K2, anti-CD40L mAb, liposome alone, or PBS and then immunized with K2 in CFA. Ten days later, lymph node T cells of the pretreated mice were cultured with APC and K2 (30 µM) for 3 days and amounts of IFN-{gamma} (a) and IL-4 (b) in the CS were quantitated.

 
Levels of IFN-{gamma} and IL-4 seen in the CS of T cells from mice pretreated with liposomal K2 and anti-CD40L mAb were almost the same as those in the CS of T cells treated with liposomal K2 alone. Recent studies demonstrated that the administration of anti-CD40L mAb altered Th1-type responses to the Th2-type responses in vivo (24, 25, 26). However, the shift of the cytokine production pattern from Th1 to Th2 was not augmented by simultaneous administration of anti-CD40L mAb in our experimental system. In addition, it was shown that administration of anti-CD40L mAb alone showed no influence on the cytokine production by the K2-primed T cells (Fig. 5Go, a and b). Almost the same low levels of IL-2 were detected in CS of the lymph node cells, irrespective of administration of liposomal peptide or PBS (data not shown). These findings suggest that the administration of liposomal K2 inhibits Th1 response but not that of Th2, and that the augmentation of the inhibitory effect of liposomal K2 by anti-CD40L mAb results from anergy induction but not from the Th2 shift.

Prevention of EAU development by pretreatment with s.c. administration of liposomal K2 and anti-CD40L mAb

It has been demonstrated that EAU is induced by Th1 cells (6, 7). We then examined whether the pretreatment with liposomal K2 and anti-CD40L mAb prevents EAU development in H-2Ak mice. Fig. 6Go shows two separate experiments. In each experiment, five mice (10 eyes) in one group were analyzed. Ten days before EAU induction by immunization with K2 in CFA, H-2Ak mice were administered s.c. either liposomal K2, anti-CD40L mAb alone, liposomal K2 together with anti-CD40L mAb, or PBS. Thereafter, the clinical score of EAU was sequentially determined. Compared with the PBS pretreatment as a control, liposomal K2 administration significantly decreased EAU clinical score (p < 0.01 at days 27–37) (Fig. 6Goa). Furthermore, additional treatment with anti-CD40L mAb augmented the inhibitory effect of liposomal K2 on EAU development, both onset and severity (Fig. 6Gob). Fig. 6Goc shows the same set of experiments as Fig. 6Gob. Although the EAU onset in the control group was detected at early stages in this particular experiment, almost complete inhibition of EAU by both anti-CD40L mAb and liposomal K2 was obtained. In contrast, pretreatment with anti-CD40L mAb alone showed no considerable influence on the EAU development as compared with that in PBS-treated mice (p > 0.1 at days 20, 23, 27, and 34) (Fig. 6God). Thus, it was demonstrated that prior administration of liposomal K2 plus anti-CD40L mAb inhibited the subsequent development of K2-induced EAU more profoundly than that of liposomal K2 alone.



View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 6. Prevention of EAU development by pretreatment with liposomal K2 or liposomal K2 plus anti-CD40L mAb. a and b, B10.BR mice were pretreated with PBS (C), liposomal K2 (P), or liposomal K2 plus anti-CD40L mAb (PA). Thereafter, the mice were immunized in the footpads and base of the tail with 100 nmol K2 in CFA. Concurrently, B. pertussis inactive bacterial suspension (1010) in a volume of 100 µl was injected intraperitoneally as an additional adjuvant. Clinical score was determined as described in Materials and Methods. c and d, B10.BR mice were pretreated with PBS (C), liposomal K2 plus anti-CD40L mAb (PA), or anti-CD40L mAb alone (A).

 
Fig. 7Go summarizes pathological scores of eye tissues from mice used for clinical assessment in the prior two experiments. Eyes were enucleated immediately after the last clinical assessment of EAU and examined histologically. Because the inflammation area of the eyes was limited, the pathological EAU was not always observed in the eye sections of positive control groups, where almost all mice showed clinically detectable EAU. Nevertheless, it seemed clear that no or few histological EAU areas were detected in liposomal K2 and liposomal K2 plus anti-CD40L groups, whereas histological EAU could be observed in substantial proportions of the eyes from PBS control group or liposome alone group (Fig. 7Go).



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 7. Pathological score of EAU. At 37 days after immunization, eyes were enucleated and histological examination was conducted as described in Materials and Methods. {circ}, Individual eyes; thick bars, average pathological score.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we showed that pretreatment of H-2Ak mice with the liposomal K2 considerably inhibited the subsequent K2-specific T cell-proliferative response and IFN-{gamma} production, but not IL-4 production. In contrast, almost the same levels of T cell-proliferative response to PPD were observed among liposomal K2-treated mice and control mice pretreated with PBS or liposome alone. Thus, the inhibition appeared to be induced in Th1 cells specific for the K2. The inhibitory effect of the liposomal K2 on the K2-specific T cell response was greater than that by K2 in IFA. Similar results were obtained in the combination of DO11.10 T cells and OVA323–339.

It was suggested that T cell tolerance by repeated s.c. injections of peptide in IFA was attributed to the T cell anergy specific for the peptide Ag (11, 13). Thus, we attempted to determine whether the T cell tolerance induced by s.c. administration of liposomal peptide was also attributed to the T cell anergy. To address this issue, we used a combination of OVA323–339 peptide and H-2Ad (BALB/c) mice that had been transferred T cells bearing DO11.10 TCR (21, 22, 23). However, Hayashi et al. (27) reported that naive DO11.10 T cells were resistant to the anergy induction. Indeed, in this experimental system, a single treatment with liposomal peptide (100 nmol) showed only a slight effect (data not shown). Then, the (DO11.10->BALB/c) mice were s.c. administered liposomal OVA323–339 (50 nmol) or OVA323–339 in IFA three times (12). Three days after the last pretreatment, these mice were immunized with OVA323–339 in CFA. The T cell responses were markedly inhibited in liposomal peptide-treated mice as compared with those treated with liposome alone (control mice) and those treated with peptide in IFA. The number of the transferred DO11.10 T cells in the draining lymph nodes of (DO11.10 -> BALB/c) mice pretreated with liposomal peptide was lower than that in those treated with peptide in IFA or liposome alone. Nevertheless, the responsiveness against OVA323–339 Ag per 5000 DO11.10 T cells in vitro was considerably decreased in the former mice pretreated with liposomal peptide as compared with that in control mice. These findings suggest that the s.c. administration of liposomal peptide induces both peptide-specific T cell, perhaps Th1, anergy, and selective death among the Th1 cells. We consider that rapid uptake and presentation of the peptide Ag in liposomes by APC may result in the engagement of TCR before sufficient expression of the costimulatory molecules such as B7 on the APC and induce anergy and/or death in the T cells (14, 15).

It has been reported that a single i.p. injection or repeated s.c. injections of peptide in IFA not only induces the peptide-specific T cell tolerance but also prevents development of autoimmune diseases in mice (11, 13). Thus, it was anticipated that liposomal peptide could be used for preventing autoimmune diseases. Indeed, the same pretreatment with liposomal K2 ameliorated EAU in the H-2Ak mice that would have been induced by immunization with K2 in CFA 10 days later.

In various autoimmune disease models, including EAE and rheumatic arthritis, i.p. administration of anti-CD40L mAb has been used to successfully prevent development of the diseases (25, 28, 29, 30, 31). However, we show herein that s.c. administration of anti-CD40L mAb (50 µg) alone exerts no influence on the development of EAU. Although it has been reported that anti-CD40L mAb functions at various sites and stages of the immune responses (32), a single s.c. administration of anti-CD40L mAb alone might no longer influence the priming of T cells by K2 in CFA administered 10 days later. This finding seems to be compatible with the result that PPD-specific T cell response (a representative Th1-type response) (33, 34, 35) was not inhibited by the administration of anti-CD40L mAb at all. H37Ra bacterial Ag including PPD was given 10 days after anti-CD40L mAb administration, and no inhibitory effect was observed in the response to PPD. Similarly, when mice were treated with anti-CD40L mAb alone, and immunized K2 in CFA 3 wk later, inhibition of the generation of the K2-specific T cell response was almost negligible. Perhaps at these stages the effect of anti-CD40L mAb no longer lasted.

Nevertheless, simultaneous s.c. administration of the same dose of anti-CD40L mAb in addition to liposomal K2 augmented the inhibitory effect of the liposomal K2 on T cell-proliferative responses and the liposomal K2-induced prevention of EAU development. Upon CD40 ligation, professional APC such as DC up-regulate costimulatory activity and efficiently activate T cells. Thus, lack of APC activation through CD40 appears to result in insufficient priming of CD4+ T cells (36, 37). Liposomal peptide Ag and anti-CD40L mAb were mixed exclusively and administered s.c. At this early stage of Ag presentation, anti-CD40L mAb blocked important costimulatory functions of APC, and T cell anergy specific for the peptide Ag in liposome might efficiently be induced. Thus, we consider that the simultaneous administration of anti-CD40L mAb and peptide Ag resulted in the synergistic induction of T cell anergy shown in this study.

Although the main mechanism underlying the augmenting effect of anti-CD40L mAb on the inhibition of disease onset and severity of EAU induced by liposomal K2 appears to be anergy induction in Th1 cells and the subsequent Th2 shift, there may be other possibilities. It has been reported that CD40 ligation activates endothelial cells and up-regulates expression of adhesion molecules (38) as well as production of proinflammatory cytokines (39). These alterations seem to promote extravasation of lymphocytes. Thus, it seems possible that anti-CD40L mAb blocks transmigration of T cells across the blood-ocular barrier and eventually inhibits development of EAU. However, this possibility appears to be unlikely, because anti-CD40L mAb may not be present at the stage of EAU development.

It has been postulated that anti-CD40L mAb promotes to shift Th1-type to Th2-type responses. In the absence of a signal through CD40, DC produce no or negligible IL-12 that results in prevention of Th1 priming necessary for developing various autoimmune diseases and allograft rejections (28, 29, 30, 31, 40, 41). However, in this study, we demonstrated that pretreatment with anti-CD40L mAb either with liposomal K2 or PBS showed no apparent influence on the productions of IFN-{gamma} and IL-4 by T cells from these mice, which were immunized with K2 in CFA 10 days after pretreatment (Fig. 5Go). Perhaps production of a Th1 cytokine, IFN-{gamma}, was inhibited sufficiently by liposomal K2 alone, and additive effects of anti-CD40L mAb might be detected only in inhibition of the T cell-proliferative responses but not apparently seen in inhibition of Th1 cytokine production in our experimental system.

In this study, we demonstrated that pretreatment with liposomal peptide and anti-CD40L mAb induced the peptide-specific T cell tolerance. We consider that our procedure would be useful for inducing Ag-specific immune suppression and preventing autoimmune diseases in which pathogenic epitope peptides are identified (42). To apply our method to clinical use, it should be pursued in further studies under what conditions T cells can be readily induced into the suppressive states following administration of liposomal peptide and anti-CD40L mAb (27).


   Footnotes
 
1 This study was supported in part by a grant-in-aid for Scientific Research by the Ministry of Health and Welfare, Japan; The Ministry of Education, Science, Sports, and Culture, Japan; and the Mochida Memorial Foundation for Medical and Pharmaceutical Research. Back

2 Current address: Department of Pathology, Shiga University of Medical Science, School of Medicine, Ohtsu, 520-2192, Japan. Back

3 Address correspondence and reprint requests to Dr. K. Onoé, Division of Immunobiology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo, 060-0815 Japan. Back

4 Abbreviations used in this paper: EAU, experimental autoimmune uveoretinitis; CD40L, CD40 ligand; CS, culture supernatant(s); EAE, experimental allergic encephalomyelitis; IRBP, interphotoreceptor retinoid-binding protein; PPD, purified protein derivative of tuberculin; RR, relative response(s); DC, dendritic cells. Back

Received for publication November 29, 1999. Accepted for publication June 21, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mochizuki, M., T. Kuwabara, C. McAllister, R. B. Nussenblatt, I. Gery. 1985. Adoptive transfer of experimental autoimmune uveoretinitis in rats: immunopathogenic mechanisms and histologic features. Invest. Opthalmol. Vis. Sci. 26:1.[Abstract/Free Full Text]
  2. Caspi, R. R., F. G. Roberge, C. G. McAllister, M. el-Saied, T. Kuwabara, I. Gery, E. Hanna, R. B. Nussenblatt. 1986. T cell lines mediating experimental autoimmune uveoretinitis (EAU) in the rat. J. Immunol. 136:928.[Abstract]
  3. Gregerson, D. S., W. F. Obritsch, S. P. Fling, J. D. Cameron. 1986. T cell lines recognize peptide fragments of S-antigen and mediate experimental autoimmune uveoretinitis and pinealitis. J. Immunol. 136:2875.[Abstract]
  4. Wacker, W. B., L. A. Donoso, C. W. Kalsow, Jr J. A. Yankeelov, D. T. Organisciak. 1977. Experimental allergic uveitis: isolation, characterization and localization of a soluble uveitopathogenic antigen from bovine retina. J. Immunol. 119:1949.[Abstract/Free Full Text]
  5. Gery, I., M. Mochizuki, R. B. Nussenblatt. 1986. Retinal specific antigens and immunopathogenic processes they provoke. Prog. Retin. Eye Res. 5:75.
  6. Caspi, R. R.. 1989. Basic mechanisms in immune-mediated uveitic disease. S. L. Lightman, ed. Immunology of Eye Disease 61. Kluwer Academic Publishers, Lancaster, U.K.
  7. Namba, K., K. Ogasawara, N. Kitaichi, N. Matsuki, A. Takahashi, Y. Sasamoto, S. Kotake, H. Matsuda, K. Iwabuchi, S. Ohno, K. Onoé. 1998. Identification of a peptide inducing experimental autoimmune uveoretinitis (EAU) in H-2Ak-carrying mice. Clin. Exp. Immunol. 111:442.[Medline]
  8. Lider, O., T. Reshef, E. Beraud, A. Ben-Nun, I. R. Cohen. 1988. Anti-idiotypic network induced by T cell vaccination against experimental autoimmune encephalomyelitis. Science 239:181.[Abstract/Free Full Text]
  9. Vandenbark, A. A., G. Hashim, H. Offner. 1989. Immunization with a synthetic T-cell receptor V-region peptide protects against experimental autoimmune encephalomyelitis. Nature 341:541.[Medline]
  10. Zaller, D. M., G. Osman, O. Kanagawa, L. Hood. 1990. Prevention and treatment of murine experimental allergic encephalomyelitis with T cell receptor V ß-specific antibodies. J. Exp. Med. 171:1943.[Abstract/Free Full Text]
  11. Gaur, A., B. Wiers, A. Liu, J. Rothbard, C. G. Fathman. 1992. Amelioration of autoimmune encephalomyelitis by myelin basic protein synthetic peptide-induced anergy. Science 258:1491.[Abstract/Free Full Text]
  12. Marusic, S., S. Tonegawa. 1997. Tolerance induction and autoimmune encephalomyelitis amelioration after administration of myelin basic protein-derived peptide. J. Exp. Med. 186:507.[Abstract/Free Full Text]
  13. Forsthuber, T., H. C. Yip, P. V. Lehmann. 1996. Induction of TH1 and TH2 immunity in neonatal mice. Science 271:1728.[Abstract]
  14. Ito, D., K. Ogasawara, K. Iwabuchi, Y. Inuyama, K. Onoé. 2000. Induction of CTL responses by simultaneous administration of liposomal peptide vaccine with anti-CD40 and anti-CTLA-4 mAb. J. Immunol. 164:1230.[Abstract/Free Full Text]
  15. Ito, D., K. Ogasawara, K. Matsushita, T. Morohashi, K. Namba, N. Matsuki, N. Kitaichi, Y. Inuyama, M. Hosokawa, E. Nakayama, et al 2000. Effective priming of cytotoxic T lymphocyte precursors by subcutaneous administration of peptide antigens in liposomes accompanied with anti-CD40 and anti-CTLA-4 antibodies. Immunobiology 201:527.[Medline]
  16. Murphy, K. M., A. B. Heimberger, D. Y. Loh. 1990. Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo. Science 250:1720.[Abstract/Free Full Text]
  17. Naruse, H., K. Ogasawara, R. Kaneda, S. Hatakeyama, T. Itoh, H. Kida, T. Miyazaki, R. A. Good, K. Onoé. 1994. A potential peptide vaccine against two different strains of influenza virus isolated at intervals of about 10 years. Proc. Natl. Acad. Sci. USA 91:9588.[Abstract/Free Full Text]
  18. Iwabuchi, C., K. Iwabuchi, K. Nakagawa, T. Takayanagi, H. Nishihori, S. Tone, K. Ogasawara, R. A. Good, K. Onoé. 1998. Intrathymic selection of NK1.1+{alpha}/ß T cell antigen receptor (TCR)+ cells in transgenic mice bearing TCR specific for chicken ovalbumin and restricted to I-Ad. Proc. Natl. Acad. Sci. USA 95:8199.[Abstract/Free Full Text]
  19. Takayanagi, T., H. Nishihori, N. Matsuki, K. Iwabuchi, K. Ogasawara, K. Onoé. 1997. Effects of non-major histocompatibility antigens on acute graft versus host reaction after allogeneic bone marrow transplantation. Bone Marrow Transplant. 20:297.[Medline]
  20. Thurau, S. R., C. C. Chan, R. B. Nussenblatt, R. R. Caspi. 1997. Oral tolerance in a murine model of relapsing experimental autoimmune uveoretinitis (EAU): induction of protective tolerance in primed animals. Clin. Exp. Immunol. 109:370.[Medline]
  21. Kearney, E. R., K. A. Pape, D. Y. Loh, M. K. Jenkins. 1994. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo. Immunity 1:327.[Medline]
  22. Pape, K. A., R. Merica, A. Mondino, A. Khoruts, M. K. Jenkins. 1998. Direct evidence that functionally impaired CD4+ T cells persist in vivo following induction of peripheral tolerance. J. Immunol. 160:4719.[Abstract/Free Full Text]
  23. Perez, V. L., L. Van Parijs, A. Biuckians, X. X. Zheng, T. B. Strom, A. K. Abbas. 1997. Induction of peripheral T cell tolerance in vivo requires CTLA-4 engagement. Immunity 6:411.[Medline]
  24. Tang, A., T. A. Judge, L. A. Turka. 1997. Blockade of CD40-CD40 ligand pathway induces tolerance in murine contact hypersensitivity. Eur. J. Immunol. 27:3143.[Medline]
  25. Stuber, E., W. Strober, M. Neurath. 1996. Blocking the CD40L-CD40 interaction in vivo specifically prevents the priming of T helper 1 cells through the inhibition of interleukin 12 secretion. J. Exp. Med. 183:693.[Abstract/Free Full Text]
  26. Hancock, W. W., M. H. Sayegh, X. G. Zheng, R. Peach, P. S. Linsley, L. A. Turka. 1996. Costimulatory function and expression of CD40 ligand, CD80, and CD86 in vascularized murine cardiac allograft rejection. Proc. Natl. Acad. Sci. USA 93:13967.[Abstract/Free Full Text]
  27. Hayashi, R. J., D. Y. Loh, O. Kanagawa, F. Wang. 1998. Differences between responses of naive and activated T cells to anergy induction. J. Immunol. 160:33.[Abstract/Free Full Text]
  28. Durie, F. H., R. A. Fava, T. M. Foy, A. Aruffo, J. A. Ledbetter, R. J. Noelle. 1993. Prevention of collagen-induced arthritis with an antibody to gp39, the ligand for CD40. Science 261:1328.[Abstract/Free Full Text]
  29. Gerritse, K., J. D. Laman, R. J. Noelle, A. Aruffo, J. A. Ledbetter, W. J. Boersma, E. Claassen. 1996. CD40-CD40 ligand interactions in experimental allergic encephalomyelitis and multiple sclerosis. Proc. Natl. Acad. Sci. USA 93:2499.[Abstract/Free Full Text]
  30. Mohan, C., Y. Shi, J. D. Laman, S. K. Datta. 1995. Interaction between CD40 and its ligand gp39 in the development of murine lupus nephritis. J. Immunol. 154:1470.[Abstract]
  31. Early, G. S., W. Zhao, C. M. Burns. 1996. Anti-CD40 ligand antibody treatment prevents the development of lupus-like nephritis in a subset of New Zealand Black x New Zealand White mice: response correlates with the absence of an anti-antibody response. J. Immunol. 157:3159.[Abstract]
  32. Grewal, I. S., R. A. Flavell. 1998. CD40 and CD154 in cell-mediated immunity. Annu. Rev. Immunol. 16:111.[Medline]
  33. Pearlman, E., J. W. Kazura, Jr F. E. Hazlett, W. H. Boom. 1993. Modulation of murine cytokine responses to mycobacterial antigens by helminth-induced T helper 2 cell responses. J. Immunol. 151:4857.[Abstract]
  34. Huygen, K., D. Abramowicz, P. Vandenbussche, F. Jacobs, J. De Bruyn, A. Kentos, A. Drowart, J. P. Van Vooren, M. Goldman. 1992. Spleen cell cytokine secretion in Mycobacterium bovis BCG-infected mice. Infect. Immun. 60:2880.[Abstract/Free Full Text]
  35. Sartono, E., Y. C. Kruize, A. Kurniawan, R. M. Maizels, M. Yazdanbakhsh. 1996. In Th2-biased lymphatic filarial patients, responses to purified protein derivative of Mycobacterium tuberculosis remain Th1. Eur. J. Immunol. 26:501.[Medline]
  36. Griggs, N. D., S. S. Agersborg, R. J. Noelle, J. A. Ledbetter, P. S. Linsley, K. S. Tung. 1996. The relative contribution of the CD28 and gp39 costimulatory pathways in the clonal expansion and pathogenic acquisition of self-reactive T cells. J. Exp. Med. 183:801.[Abstract/Free Full Text]
  37. Howard, L. M., A. J. Miga, C. L. Vanderlugt, M. C. Dal Canto, J. D. Laman, R. J. Noelle, S. D. Miller. 1999. Mechanisms of immunotherapeutic intervention by anti-CD40L (CD154) antibody in an animal model of multiple sclerosis. J. Clin. Invest. 103:281.[Medline]
  38. Hollenbaugh, D., N. Mischel-Petty, C. P. Edwards, J. C. Simon, R. W. Denfeld, P. A. Kiener, A. Aruffo. 1995. Expression of functional CD40 by vascular endothelial cells. J. Exp. Med. 182:33.[Abstract/Free Full Text]
  39. Dechanet, J., C. Grosset, J. L. Taupin, P. Merville, J. Banchereau, J. Ripoche, J. F. Moreau. 1997. CD40 ligand stimulates proinflammatory cytokine production by human endothelial cells. J. Immunol. 159:5640.[Abstract]
  40. Niimi, M., T. C. Pearson, C. P. Larsen, D. Z. Alexander, D. Hollenbaugh, A. Aruffo, P. S. Linsley, E. Thomas, K. Campbell, W. C. Fanslow, et al 1998. The role of the CD40 pathway in alloantigen-induced hyporesponsiveness in vivo. J. Immunol. 161:5331.[Abstract/Free Full Text]
  41. Parker, D. C., D. L. Greiner, N. E. Phillips, M. C. Appel, A. W. Steele, F. H. Durie, R. J. Noelle, J. P. Mordes, A. A. Rossini. 1995. Survival of mouse pancreatic islet allografts in recipients treated with allogeneic small lymphocytes and antibody to CD40 ligand. Proc. Natl. Acad. Sci. USA 92:9560.[Abstract/Free Full Text]
  42. Haneji, N., T. Nakamura, K. Takio, K. Yanagi, H. Higashiyama, I. Saito, S. Noji, H. Sugino, Y. Hayashi. 1997. Identification of {alpha}-fodrin as a candidate autoantigen in primary Sjögren’s syndrome. Science 25:604.



This article has been cited by other articles:


Home page
 J. Immunol.Home page
M. Kitamura, K. Iwabuchi, N. Kitaichi, S. Kon, H. Kitamei, K. Namba, K. Yoshida, D. T. Denhardt, S. R. Rittling, S. Ohno, et al.
Osteopontin Aggravates Experimental Autoimmune Uveoretinitis in Mice
J. Immunol., May 15, 2007; 178(10): 6567 - 6572.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
H. Kitamei, K. Iwabuchi, K. Namba, K. Yoshida, Y. Yanagawa, N. Kitaichi, M. Kitamura, S. Ohno, and K. Onoe
Amelioration of experimental autoimmune uveoretinitis (EAU) with an inhibitor of nuclear factor-{kappa}B (NF-{kappa}B), pyrrolidine dithiocarbamate
J. Leukoc. Biol., June 1, 2006; 79(6): 1193 - 1201.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
N. Hashida, N. Ohguro, K. Nakai, M. Kobashi-Hashida, S.-i. Hashimoto, K. Matsushima, and Y. Tano
Microarray Analysis of Cytokine and Chemokine Gene Expression after Prednisolone Treatment in Murine Experimental Autoimmune Uveoretinitis
Invest. Ophthalmol. Vis. Sci., November 1, 2005; 46(11): 4224 - 4234.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
N. Kitaichi, S. Kotake, T. Morohashi, K. Onoe, S. Ohno, and A. W. Taylor
Diminution of experimental autoimmune uveoretinitis (EAU) in mice depleted of NK cells
J. Leukoc. Biol., December 1, 2002; 72(6): 1117 - 1121.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. Stienekemeier, K. Falk, O. Rotzschke, A. Weishaupt, C. Schneider, K. V. Toyka, R. Gold, and J. L. Strominger
Vaccination, prevention, and treatment of experimental autoimmune neuritis (EAN) by an oligomerized T cell epitope
PNAS, November 20, 2001; 98(24): 13872 - 13877.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Namba, K.
Right arrow Articles by Onoé, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Namba, K.
Right arrow Articles by Onoé, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS