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(Journal of Leukocyte Biology. 2001;69:741-746.)
© 2001 by Society for Leukocyte Biology

Blood mononuclear cells induce regulatory NK T thymocytes in anterior chamber-associated immune deviation

Yafei Wang*,{dagger}, Irving Goldschneider*, James O’Rourke*,{dagger} and Robert E. Cone*,{dagger}

* Department of Pathology and
{dagger} Vision Immunology Center, University of Connecticut Health Center, Farmington

Correspondence: Robert E. Cone, Ph.D., Department of Pathology, School of Medicine, The University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030-3105. E-mail: Cone{at}idx.uchc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Injection of antigen into the anterior chamber (AC) of the eye, an immunologically privileged site, is associated with the induction of immune deviation, as evidenced by T helper cell (Th) 1 to Th2 cell polarization. We recently demonstrated that AC-associated immune deviation (ACAID) is a thymus-dependent phenomenon initiated by the formation of regulatory {alpha},ß T-cell receptor-positive CD4- CD8- thymocytes (THYregs). In this study, the afferent and efferent limbs of this immunoregulatory loop were traced from peripheral blood to the thymus and then to the spleen by adoptive-transfer assays. The results demonstrate that (1) F4/80+ CD1+ peripheral blood mononuclear cells from mice whose ACs were injected with trinitrophenol-bovine serum albumin induce the appearance of natural killer (NK) 1.1+ THYreg in naïve recipients within 24 h of intravenous infusion; (2) these NK THYregs induce (or generate) suppressor-effector T cells in the spleens of adoptive recipients; (3) these suppressor-effector spleen cells, but not the NK THYregs themselves, directly inhibit the expression of delayed-type hypersensitivity in sensitized recipients; and (4) peripheral blood mononuclear cells from AC-injected mice do not induce ACAID in thymectomized recipients. These results confirm our hypothesis that ACAID is a model of centrally induced dominant tolerance mediated by CD-1-dependent NK T cells of recent thymic origin. The results also provide evidence of a novel tolerance induction pathway by which blood-borne antigen-presenting cells generated by antigen injection into an immunologically privileged site transport antigen to the thymus and induce the formation and export of THYreg.

Key Words: NK T cells • thymus • immunoregulation • immune deviation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antigen-specific regulation of T-lymphocyte responses in the peripheral lymphoid tissues may be manifested as a cytokine-directed diversion of cell-mediated to antibody-mediated immunity [1 , 2 ]. One of the routes by which so-called immune deviation can be induced involves certain "immunologically privileged" sites, such as the eye. For example, the introduction of an antigen into the anterior chamber (AC) of the eye induces the systemic production of antigen-specific immunoglobulin (Ig) M and IgG1 antibodies and the suppression of delayed-type hypersensitivity (DTH) and IgG2 antibody production in mice [3 , 4 ]. This phenomenon of anterior-chamber-associated immunological deviation (ACAID) is mediated by CD4+ and CD8+ suppressor-effector T cells that inhibit the induction and the expression of DTH, respectively [5 6 7 ]. The intravenous (i.v.) transfer of F4/80+ peripheral blood mononuclear cells (PBMCs) derived from the iris and ciliary body of the AC-injected eye can induce the appearance of CD4+ and CD8+ suppressor-effector T cells in the spleen [4 , 8 ]. In addition, B cells have been shown to serve as intermediaries in the activation of spleen-immunoregulatory T cells [9 ].

We have recently demonstrated that an intact thymus is required for the induction of ACAID in AC-injected mice and that {alpha},ß T-cell receptor (TCR)-positive (TCR+) CD4- CD8- thymocytes (THYs) from these mice can transfer suppression of DTH to antigen-primed or naïve recipient mice [10 ]. These observations suggested that the thymus is the initial lymphoid site involved in the induction of ACAID. We therefore reasoned that the PBMCs that transfer ACAID [4 ] may do so by migrating to the thymus and inducing the formation of immunoregulatory THYs (THYregs), which subsequently migrate to the spleen.

In this report, we demonstrate that in ACAID the THYregs expressed the natural killer (NK) 1.1 marker and that their formation was induced by F4/80+ PBMCs from CD1+ AC-injected donors (AC-PBMCs). After i.v. infusion, these THYregs established ACAID by suppressing the induction but not the expression of DTH, possibly by means of induction and/or formation of suppressor-effector T cells in the spleen. However, the AC-PBMCs that induced these NK THYregs were unable to induce ACAID in thymectomized (Tx) mice. Hence, these results suggest a heretofore undescribed route of acquired tolerance induction by which blood-borne antigen-presenting cells (APCs), presumably from an immunologically privileged site, transport non-self antigen to the thymus and present it in a manner that leads to the formation and export of NK THYregs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Female BALB/c, BALB/c CD1-/-, and C56BL/6 x BALB/c (CB6) F1 mice, 5 to 6 weeks old, were purchased from River Run Breeders of Harlan Laboratory (Indianapolis, IN) or Jackson Laboratory (Bar Harbor, ME). All animals were maintained by the Center for Laboratory Animal Care at the University of Connecticut Health Center.

Antigens
2,4,6-Trinitrobenzenesulfonic acid and bovine serum albumin (BSA) were obtained from Sigma Chemical Co. (St. Louis, MO). 2-Chloro-5-triphtane [picryl chloride (PCl)] was obtained from Chemica Alta Ltd. (Edmonton, Alberta, Canada). Trinitrophenol (TNP)-BSA (10-mg/mL solution) was prepared by mixing 500 mg of BSA and 2,4,6-trinitrobenzenesulfonic acid in 50 mL of 0.1 M Na2CO3. The solution was stirred overnight at room temperature and then dialyzed against 1 L of 0.01 M NaHCO3.

Thymectomy
Five-week-old female mice were Tx by suction, as described previously [10 ].

AC injection
An oblique transcorneal paracentesis was performed under microscopic control with a 33-gauge needle, and 5 µL of TNP-BSA (50 µg) or BSA (50 µg) were injected [10 ].

Elicitation of contact DTH
Mice were sensitized systemically by intradermal injection of 0.2 mL of TNP-BSA (100 µg) with complete Freund’s adjuvant (Sigma) into the abdominal region and were challenged 7 days later by epicutaneous application (15 µL) of 1% PCl [in acetone-olive oil (4:1)] to the right ear or footpad. The DTH response was determined by measuring ear or footpad swelling with an engineer’s micrometer (Mitatoyo Mfg., Tokyo, Japan) 24 h after challenge. Swelling was determined by the difference in DTH units (1 DTH unit = 2.54 x 103 mm) between the challenged right ear or footpad and the unchallenged left ear or footpad of experimental animals. Swelling is expressed in micrometers. Results were corrected for nonspecific swelling by subtracting the difference in DTH units between the ears or footpads of unsensitized control mice challenged unilaterally with PCl as described previously [10 ]. To ensure consistency and objectivity, these measurements were made by a single individual in a blinded manner so that the experimental group to which each animal belonged was not known in advance.

Immunomagnetic separation of THY subsets
Suspensions of thymus cells, prepared 24 h after injection of TNP-BSA into ACs, were first incubated with purified anti-mouse NK1.1 monoclonal antibody (10µL/107 cells) (PharMingen, San Diego, CA) for 15 min at 6–12°C and then incubated with superparamagnetic microbeads (10µL/107 cells) conjugated with polyclonal goat anti-mouse immunoglobulin G (IgG) antibodies (Miltenyi Biotec GmbH, Sunnyvale, CA). The cells were then washed twice in phosphate-buffered saline (PBS) (pH 7.2) containing 1% BSA, 0.01% sodium azide, and 5 mM ethylenediaminetetraacetate, and 1.2 x 108 cells in 1 mL of buffer were applied to a separation column (type BS, Vario MACS; Miltenyi Biotec GmbH). The negative and positive fractions were collected as previously described [10 ] and injected i.v. into recipient mice. Results were compared with those obtained with aliquots of antibody-treated and non-antibody-treated, unseparated controls.

Adoptive-transfer assays
THY
Twenty-four hours after injection of TNP-BSA into the ACs of nonsensitized mice, thymuses were removed, freed of adherent lymph nodes, and gently disrupted in RPMI 1640 culture medium. For i.v. transfer, 1.2 x 107 AC THYs in 0.5 mL of PBS were injected into the tail vein of TNP-BSA-sensitized mice. For transfer into the ear pinna, 5.65 x 105 AC-THYs in 10 µL were injected subcutaneously (s.c.) immediately before and at the site of epicutaneous application of PCl. In control experiments, THYs were obtained from non-AC-injected, nonsensitized mice.

Spleen cells
Seven days after AC injection or i.v. injection of AC-THYs into sensitized mice, spleens were removed, diced, and expressed through a steel mesh into RPMI 1640 medium. Spleen cells (SPLs) (5 x 105) from AC-injected mice or mice receiving AC-THYs (AC-SPLs) in PBS were injected s.c. into the right ear pinna of TNP-BSA-sensitized or naïve mice immediately prior to and at the site of challenge by epicutaneous application of PCl.

PBMCs
PBMCs were prepared as described elsewhere [4 ]. Briefly, blood leukocytes from groups of five to seven mice were collected by centrifugation 24 h after injection of TNP-BSA in the mouse AC (AC-PBMCs), and the cells were layered over Lymphoprep medium (Cardinal Associates, Inc. Santa Fe, NM) and centrifuged 20 min at 500 rpm. The PBMCs at the interface were collected, washed twice with PBS, and injected i.v. (2.5 x 106 in 0.5 mL) into TNP-BSA-sensitized or naïve mice. In some experiments, 2 x 106 PBMCs were treated with anti-F4/80 antibody (10 µg) or normal rat IgG2b (PharMingen) in 200 µL of PBS containing 10 mM ethylenediaminetetraacetate for 15 min at 6–12°C prior to i.v. transfer into TNP-BSA-sensitized mice.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
AC-THYs inhibit the induction but not the expression of DTH in adoptive recipients
We previously demonstrated that AC-THYs can transfer ACAID to mice sensitized with the homologous antigen within 7 days after i.v. injection [10 ]. However, we did not determine whether AC-THYs inhibit the expression of DTH directly or indirectly. Toward this end, AC-THYs were injected either i.v. into sensitized recipients 7 days before challenge of the ear pinna with PCl or s.c. into the ear pinna at the time and site of challenge with PCl.

As shown in Figure 1 , AC-THYs were able to suppress the induction of DTH when transferred systemically, but they were unable to suppress the expression of DTH when injected locally. In contrast, SPLs from sensitized mice that had been injected i.v. with AC-THYs 1 week previously directly suppressed DTH when SPLs were injected s.c. at the site of challenge (Fig. 2 ). These results suggest that AC-THYs induce the formation of and/or differentiate into suppressor-effector T cells in the spleen within 1 week after i.v. transfer.



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  		Figure 1. AC-THYs indirectly suppress DTH. THYs (1.2 x 107) from naïve mice (naïve THY) or from mice whose ACs were with TNP-BSA 1 day previously (AC-THYs) were transferred i.v. into TNP-BSA-sensitized recipients. One week later, the recipients were challenged with epicutaneous PCl. Another group of sensitized recipients was injected s.c. with naïve or AC-THYs (5.7 x 105) in the ear pinna at the site and time of challenge with PCl. In both instances, ear swelling was measured 24 h after challenge. Control animals are designated DTH, TNP-BSA-sensitized mice challenged with PCl and ACAID, AC-injected, TNP-BSA-sensitized mice challenged with PCl. Results are representative of data from two experiments. Values are means ± SE of ear swelling measurements for three mice per group (see Materials and Methods).

 


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  		Figure 2. AC-THYs enable SPLs to directly suppress DTH. SPLs (106) from naïve mice (naïve SPLs) or from groups of TNP-BSA-sensitized mice whose ACs had been injected with TNP-BSA (AC-SPLs) or had been injected i.v. with naïve THYs or AC-THYs 1 week previously were transferred s.c. into the ear pinna of TNP-BSA-sensitized recipients concurrently with an epicutaneous challenge of PCl. ACAID and DTH controls were included as in Figure 1 . The results are representative of data obtained in two experiments. Data are means ± SE of ear swelling measurements (in micrometers) from three mice per group.

 
Immunoregulatory AC-THYs are NK1.1+
The AC-THYs that transfer ACAID to recipient mice have an {alpha},ß TCR+ CD4- CD8- phenotype [10 ]. Because many immunoregulatory cells having this phenotype are NK T cells [11 , 12 ], we used immunomagnetic separation to determine whether the immunoregulatory THYs obtained 24 h after AC injection expressed the NK1.1 marker. CB6 mice were used in these experiments to detect NK1.1. DTH was determined by measuring hind footpad swelling only, because technical difficulties were encountered in determining ear swelling in these mice. The results in Figure 3 show that 4.5 x 106 NK1.1+ AC-THYs reduced DTH in sensitized recipients to the same extent as 1.3 x 107 unfractionated AC-THYs (p > 0.1). In contrast, 1.25 x 107 NK1.1- AC-THYs did not transfer suppression of DTH.



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  		Figure 3. Regulatory AC-THYs are NK1.1+. THYs from CB6 mice whose ACs were injected with TNP-BSA 24 h previously (AC-THYs) were incubated with anti-NK1.1 antibody and subjected to immunomagnetic separation. Totals of 1.3 x 107 nonfractionated AC-THYs, 4.5 x 106 effluent (NK1.1+), and 1.25 x 107 eluate (NK1.1-) AC-THYs were injected i.v. into TNP-BSA-sensitized mice. One week later, the hind footpad was challenged with epicutaneous PCl, and swelling was measured 24 h later. The data are means ± SE of ear swelling measurements (in micrometers) from three mice per group.

 
AC-PBMCs induce the appearance of THYregs in adoptive recipients
AC-PBMCs have been shown to transfer ACAID when injected iv. into adoptive recipients [4 ]. Inasmuch as AC-THYs also can transfer ACAID [10 ], as noted in our preceding results, it was of interest to determine whether these two phenomena were related. PBMCs that were obtained 24 h after injection of TNP-BSA into ACs were transferred i.v. into naïve mice, and 24 h later THYs from these primary recipients were injected i.v. into TNP-BSA-sensitized mice. One week later, these secondary recipients were challenged with PCl, and ear swelling was measured 24 h after the challenge. The results were compared with those obtained after i.v. injection of AC-PBMCs directly into sensitized mice. As shown in Figure 4 , DTH was suppressed in mice receiving either AC-PBMCs or THYs from recipients of AC-PBMCs. However, DTH was not suppressed in mice receiving naïve PBMCs or THYs from recipients of naïve PBMCs.



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  		Figure 4. PBMCs from AC-injected mice induce THYregs. PBMCs from mice whose ACs were injected with TNP-BSA 48 h previously (AC-PBMCs) or from PBMC-naïve mice were transferred i.v. into PBMC-naïve recipients. One day later, THYs were obtained from these primary recipients and injected i.v. into TNP-BSA-sensitized recipients. Seven days later, the ears of these secondary recipients were challenged with epicutaneous PCl. Results were compared with those obtained in sensitized primary recipients of naïve PBMCs or AC-PBMCs. Data are means ± SE of ear swelling measurements (in micrometers) from six mice per group.

 
We have previously demonstrated that regulatory T cells induced by AC injection appear in the thymus before they are seen in the spleen and that the appearance of regulatory AC-SPL cells is prevented by prior thymectomy [10 ]. Similarly, in the present study, AC-PBMCs did not induce ACAID when they were injected i.v. into TNP-BSA-sensitized mice 1 week after thymectomy (data not shown).

AC-PBMCs that induce THYregs express an F4/80+ CD1+ phenotype
As reported elsewhere [4 , 8 ] and confirmed here (data not shown), the PBMCs that transfer ACAID to sensitized recipients express F4/80, a selective marker for monocytes/macrophages and dendritic cells (DCs). To determine whether the AC-PBMCs that induce the appearance of THYregs also express F4/80, AC-PBMCs were incubated with anti-F4/80 antibody or normal (control) rat IgG and injected into TNP-BSA-sensitized mice. As shown in Figure 5 , the anti-F4/80 antibody-treated AC-PBMCs failed to induce THYregs.



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  		Figure 5. F4/80+ AC-PBMCs induce THYregs. AC-PBMCs from mice injected with TNP-BSA 24 h previously were incubated with anti-F4/80 IgG or normal rat IgG and introduced by i.v. injection into TNP-BSA-sensitized mice. Twenty-four hours after injection of AC-PBMCs, 106 THYs from these primary recipients were injected i.v. into TNP-BSA-sensitized recipients. Seven days later, the footpads of these secondary recipients were challenged with PCl, and swelling was measured 24 h later. The data are average footpad swelling ± SE (in micrometers) of three to six mice per group.

 
Because antigen recognition by NK T cells is restricted by CD1 [13 ], we determined whether AC-PBMCs from CD1-/- mice were able to transfer ACAID. As shown in Figure 6 , PBMCs from AC-injected CD1-/- mice did not transfer ACAID to CD1+/+ mice. Moreover, DTH was not suppressed in NK T-cell-deficient CD1-/- mice that received AC-PBMCs from CD1+/+ mice.



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  		Figure 6. AC-PBMC from CD1-/- mice do not induce suppression of DTH in CD1+/+ mice. PBMCs obtained from CD1-/- and CD1+/+ BALB/c mice 48 h after injection into their ACs were infused i.v. into TNP-BSA-sensitized CD1+/+ or CD-/- BALB/c mice, respectively. One week later, the recipients were challenged with epicutaneous PCl. Data are means ± SE of ear swelling measurements (in micrometers) from three mice per group.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our observation that AC-THYregs are NK1.1+ is consistent with the recent observations by Sonoda et al. [14 ] that the induction of ACAID is dependent on NK T cells. Indeed, our previous demonstration [10 ], which showed that thymectomy prevents the induction of ACAID and that regulatory cells disappear from the thymus within 4 days after AC injection of antigen, strongly suggests that the immunoregulatory NK T cells in the spleen [14 ] are recent thymic emigrants (RTEs). Such a scenario was anticipated several decades ago in the initial description of "spleen-seeking" THYregs [15 , 16 ]. Although it has been suggested that the splenic NK T cells function as suppressor-inducer cells [14 ], this has not been established experimentally. Even the present demonstration that AC-SPLs, but not AC-THYs, can directly suppress the expression of DTH does not exclude the possibility that the AC-THYs subsequently develop into suppressor-effector T cells. Thus, it has been demonstrated that most RTEs undergo further differentiation during their first week in the peripheral lymphoid tissues [17 ] and that antigen can polarize CD-1-reactive NK T cells toward Th2 cytokine production [18 , 19 ], the dominant profile in ACAID [20 , 21 ]. Furthermore, both CD4+ and CD8+ subsets of NK T cells have been described [22 ]. To resolve this issue, adoptive-transfer experiments using Ly 45 congenic mice are in progress to trace the phenotypic and functional development of NK1.1+ AC-THY in spleens of sensitized recipients.

It has previously been shown that F4/80+ macrophages derived from the iris and ciliary body of the eye migrate via the blood to the spleen, where they are presumed to act as APCs that induce the development THYregs [4 , 5 ]. In the eye, these macrophages (or DCs) are exposed to a variety of immunosuppressive factors, including transforming growth factor ß (TGF-ß) [23 ], which can down-regulate interleukin (IL)-12 expression [24 ] and possibly can up-regulate CD-1 expression [14 ]. The present results show that F4/80+ PBMCs from AC-injected CD1+/+ but not CD-/- mice rapidly induced the appearance of THYreg when injected i.v. into naïve recipients. Furthermore, the results demonstrate that AC-PBMCs from CD1+/+ mice were unable to induce ACAID in CD1-/- recipients, which lack NK T cells [25 ]. This is consistent with the failure of CD1-/- mice to develop ACAID after injection of antigen into the ACs [14 ].

Hence, we postulate that there exists an efficient mechanism by which antigen is transported from the eye to the thymus, where it is presented in a manner that preferentially selects and activates the NK1.1+ population of {alpha},ß TCR double-negative THYs. Indeed, our results suggest that the blood-thymus route of APC migration is essential for the induction of ACAID, because AC-PBMCs cannot transfer ACAID to Tx recipients. Thus, although AC-PBMCs might be necessary for the generation of NK THYreg RTEs, they may not be required for the development of effector-suppressor cells in the spleen. Nonetheless, the possibility of an important role for F4/80+ APCs of ocular origin in enhancing, localizing, and/or modulating the production of THYregs in the spleen, possibly in concert with splenic B cells [9 ], cannot be excluded [26 ]. Indeed, it is possible that different populations of F4/80+ APCs are responsible for transporting antigen to the thymus and the spleen in ACAID. Thus, for example, peritoneal exudate cells pulsed in vitro with antigen and TGF-ß seem to be able to directly activate CD8+ splenic regulatory T cells [27 ], possibly by means of the delivery of IL-10 and antigen [28 ]. Alternatively, thymus-seeking IL-10-secreting APCs may promote the activation of thymic NK T cells, which themselves may produce IL-10 and/or TGF-ß upon migration to the spleen [29 ]. Under these circumstances, the F4/80+ APCs that migrate to the spleen may provide a second exposure of antigen to the NK T-cell RTEs. A schematic representation of these relationships is shown in Figure 7 .



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  		Figure 7. Schematic representation of the ocular-thymic-splenic immunoregulatory loop in ACAID. Antigen injected into the AC of the eye is processed by APCs from the iris and ciliary bodies (APC-ICBs) in the presence of immunoregulatory cytokines (e.g., TGF-ß). These immunoregulatory APCs then migrate to the blood, from which they presumably traffic to the thymus and spleen. In the thymus, they selectively activate {alpha},ß TCR+ NK1.1+ double-negative THYs, resulting in the generation and export of immunoregulatory NK T cells. After migrating to the spleen, these NK RTEs either induce the formation of or differentiate into Th2-type effector-suppressor T cells. Other APC-ICBs that migrate directly to spleen may influence this process, as may antigen-presenting B lymphocytes (see Discussion).

 
In addition to their requirement in ACAID, NK THYregs have been implicated in the induction of systemic tolerance in a variety of autoimmune, allotransplantation, and graft-versus-host disorders [30 31 32 ]. They also appear to represent a major subset of neonatal suppressor cells [33 ]. In many instances, these NK THYregs are suspected to be RTEs, because their appearance in the periphery closely follows either intrathymic (i.t.) injection (or transgenic expression) of antigen in the thymus or thymic transplantation and is prevented by timed thymectomy [34 35 36 37 38 ]. It is of special interest, therefore, that tolerance to experimental allergic encephalomyelitis can be transferred by i.v. injection of thymic DCs from rats injected i.t. with myelin basic protein [39 ] and that the establishment of such tolerance depends on the presence of an intact thymus. This suggests that these tolerogenic DCs may have to return to the host thymus to be effective.

To our knowledge, the present study is the first to demonstrate the ability of blood-borne APCs to induce immunological deviation by a thymus-dependent route. It, of course, remains to be demonstrated directly that these APCs: (1) arise from the eye and (2) physically transport antigen to the thymus. It will also be of interest to determine whether they appear after the injection of antigen into immunologically privileged sites other than the eye. However, it is unlikely that this phenomenon is restricted to APCs from immunologically privileged sites, given that thymus-seeking APCs may have a role in the establishment of peripheral tolerance in normal adult rats that are given footpad injections of ovalbumin in CF antigen ([40 ] and in recipients of cardiac allografts given monocyte-derived DC precursors [41 , 42 ]. Inasmuch as antigen-pulsed DCs and/or macrophages can initiate or maintain peripheral tolerance [39 , 41 , 43 , 44 ], especially when exposed to regulatory cytokines [27 , 28 , 45 46 47 48 ], it will be important to determine whether tolerogenic APCs similar to those observed in ACAID can be generated (or expanded) ex vivo. Reinfusion experiments could then explore the feasibility of establishing acquired thymic tolerance without the need for ACs or i.t. injection.


   ACKNOWLEDGEMENTS
 
This research was supported by American Heart Association grant 9750851A, the Connecticut Lions Eye Research Foundation, and the National Eye Institute, U.S.P.H.S. grant EY13243.

We gratefully thank Ruth Faasen and Catherine Mitchell for their assistance in the preparation of the manuscript and Dr. H. Leo Aguila for his advice.

Received October 8, 2000; revised November 27, 2000; accepted November 28, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Rocken, M., Shevach, E. M. (1996) Immune deviation—the third dimension of non-deletional tolerance Immunol. Rev. 149,175-194[Medline]
  2. Aebischer, I., Stadler, B. M. (1996) TH1-TH2 cells in allergic responses: at the limits of a concept Adv. Immunol 61,341-403[Medline]
  3. Streilein, J. W. (1987) Immune regulation and the eye: a dangerous compromise FASEB J 1,199-208[Abstract]
  4. Wilbanks, G. A., Mammolenti, M., Streilein, J. W. (1991) Studies on the induction of anterior chamber-associated immune deviation (ACAID). II. Eye-derived cells participate in generating blood-borne signals that induce ACAID J. Immunol. 146,3018-3024[Abstract]
  5. Wilbanks, G. A., Streilein, J. W. (1990) Characterization of suppressor cells in anterior chamber-associated immune deviation (ACAID) induced by soluble antigen. Evidence of two functionally and phenotypically distinct T suppressor cell populations Immunology 71,383-389[Medline]
  6. Ferguson, T. A., Waldrep, J. C., Kaplan, H. J. (1987) The immune response and the eye: the nature of T suppressor cell induction in anterior chamber-associated immune deviation (ACAID) J. Immunol. 139,352-357[Abstract]
  7. Streilein, J. W., Niederkorn, J. Y. (1981) Induction of anterior chamber-associated immune deviation requires an intact, functional spleen J. Exp. Med. 153,1058-1067[Abstract/Free Full Text]
  8. Wilbanks, G. A., Streilein, J. W. (1992) Macrophages capable of inducing anterior chamber associated immune deviation demonstrate spleen-seeking migratory properties Reg. Immunol. 4,130-137[Medline]
  9. D’Orazio, T. G., Niederkorn, J. Y. (1995) Splenic B cells are required for tolerogenic antigen presentation in the production of anterior chamber-associated immune deviation J. Immunol. 160,2089-2098[Abstract/Free Full Text]
  10. Wang, Y., Goldschneider, I., Foss, D., Wu, D. Y., O’Rourke, J., Cone, R. E. (1997) Direct thymic involvement in anterior chamber-associated immune deviation J. Immunol. 158,2150-2155[Abstract]
  11. Bendelac, A., Rivera, M. N., Park, S. H., Roark, J. H. (1997) Mouse CD1-specific NK1 T cells: development, specificity, and function Annu. Rev. Immunol. 15,535-562[Medline]
  12. Strober, S., Cheng, L., Palathumy, Dejbaksh, -Jones, S., Huie, P., Sibloy, R. (1996) Double negative (CD4-, CD8-, a,b+) T cells which promote tolerance induction and regulate autoimmunity Immunol. Rev. 149,217-230[Medline]
  13. Bendelac, A., Lantz, O., Quinby, M. E., Yewdell, J. W., Bennink, J. R., Brutkiewicz, R. R. (1995) CD1 recognition by mouse NK1+ T lymphocytes Science 268,863-865[Abstract/Free Full Text]
  14. Sonoda, K.-H., Exley, M., Snapper, S., Balk, S. T., Stein-Streilein, J. (1999) CD1-reactive natural killer T cells are required for development of systemic tolerance through a privileged site J. Exp. Med. 180,1215-1226
  15. Gershon, R. K., Lance, E. M., Kondo, K. (1974) Immunoregulatory role of spleen-localizing thymocytes J. Immunol. 112,546-554[Abstract/Free Full Text]
  16. Wu, C.-Y., Lance, E. M. (1974) Immunoregulation by spleen seeking thymocytes. II Role in the response to sheep erythrocytes Cell. Immunol. 13,1-11[Medline]
  17. Hosseinzadeh, H., Goldschneider, I. (1992) Recent thymic emigrants in the rat express a unique antigenic phenotype and undergo post-thymic maturation in peripheral lymphoid tissue J. Immunol. 150,1670-1679[Abstract]
  18. Burdin, N., Brossay, L., Kronenberg, M. (1999) Immunization with a-galactosylceramide polarizes CD-1 reactive NK T cells towards Th2 cytokine synthesis Eur. J. Immunol. 29,2014-2025[Medline]
  19. Sonoda, K.-H., Faunce, D. E., Taniguchi, M., Stein-Streilein, J. (2000) NK T cell derived IL-10 is essential for ACAID Keystone Symp. Mechanisms Immunol. Tolerance Breakdown ,70 Keystone, CO.
  20. Kosiewicz, M. M., Alard, P., Streilein, J. W. (1998) Alterations in cytokine production following intraocular injection of soluble protein antigen: Impairment in IFN-{alpha} and induction of TGF-ß and IL-4 production J. Immunol. 161,5382-5390[Abstract/Free Full Text]
  21. Li, X.-Y., D’Orazio, T., Niederkorn, J. Y. (1996) Role of Th1 and Th2 cells in anterior chamber-associated immune deviation Immunology 89,34-40[Medline]
  22. Hammond, K. J., Pelikan, S. B., Crowe, N. Y., Randle-Barrett, E., Nakayama, T., Taniguchi, M., Smyth, M. J., van Driel, I. R., Scollay, R., Baxter, A. G., Godfrey, D. I. (1999) NKT cells are phenotypically and functionally diverse Eur. J. Immunol. 29,3768-3781[Medline]
  23. Wilbanks, G. A., Mammolenti, M., Streilein, J. W. (1992) Studies on the induction of anterior chamber-associated immune deviation (ACAID). III. Induction of ACAID depends upon intraocular transforming growth factor-beta Eur. J. Immunol. 22,165-173[Medline]
  24. Bright, J. J., Sriram, S. (1998) TGF-ß inhibits IL-12-induced activation of Jak-STAT pathway in T lymphocytes J. Immunol. 161,1772-1777[Abstract/Free Full Text]
  25. Chen, Y.-H., Chiu, N. M., Mandal, M., Wang, N., Wang, C. R. (1997) Impaired NK1+ T cell development and early IL-4 production in CD1-deficient mice Immunity 6,459-467[Medline]
  26. Faunce, D. E., Sonoda, K.-H., Stein-Streilein, J. (2000) MIP-2 mediated recruitment of NK T cells to the spleen is required for the induction of systemic tolerance Keystone Symp. Mechanisms Immunol. Tolerance Breakdown ,76 Keystone, CO.
  27. Kezuka, T., Streilein, J. W. (2000) In vitro generation of regulatory CD8+ T cells similar to those found in mice with antigen chamber-associated immune deviation Invest. Ophthalmol. Vis. Sci. 41,1803-1811[Abstract/Free Full Text]
  28. D’Orazio, T., Neiderkorn, J. Y. (1998) A novel role for TGF-ß and IL-10 in the induction of immune privilege J. Immunol. 160,2089-2098
  29. Stein-Streilein, J., Sonoda, K.-H., Faunce, D. J., Zang-Hoover, J. (2000) Regulation of adoptive immune responses by innate cells expressing NK markers and antigen transporting macrophages J. Leukoc. Biol. 67,488-494[Abstract]
  30. Mieza, M. A., Itoh, T., Cui, J. Q., Makina, Y., Kawgno, T., Tsuchida, T., Koike, T., Shira, T., Yagita, H., Matsuzawa, A., Koseki, H., Taniguchi, M. (1996) Selective reduction of V alpha 14+ NK T cells associated with disease development in autoimmune-prone mice J. Immunol. 156,4035-4040[Abstract]
  31. Hammond, K., Poulton, L., Palimsano, P., Silverira, P., Godfrey, D., Baxter, G. (1998) Alpha/beta-T cell receptor (TCR)+ CD4- CD8- (NKT) thymocytes prevent insulin-dependent diabetes mellitus in nonobese diabetic (NOD)/Lt mice by the influence of interleukin (IL)-4 and/or IL-10 J. Exp. Med. 197,1047-1056
  32. Zeng, D., Lewis, D., Dejbaksh-Jones, S., Lan, F., Garcia-Ojeda, S. M., Sibley, M., Strober, S. (1999) Bone marrow NK1.1- and NK1.1+ T cells reciprocally regulate acute graft versus host disease J. Exp. Med. 189,1073-1081[Abstract/Free Full Text]
  33. Hammond, K., Cain, W., Van Driel, I., Godfrey, D. (1998) Three day neonatal thymectomy selectively depletes NK1.1+ T cells Int. Immunol. 10,1491-1499[Abstract/Free Full Text]
  34. Khoury, S. J., Sayegh, M. H., Hancock, W. W., Gallon, L., Carpenter, C. B., Weiner, H. L. (1993) Acquired tolerance to experimental autoimmune encephalomyelitis by intrathymic injection of myelin basic protein or its major encephalitogenic peptide J. Exp. Med. 178,559-566[Abstract/Free Full Text]
  35. Posselt, A. M., Barker, C. F., Tomaszewiski, J. E., Markmann, J. F., Choti, M. A., Naji, A. (1990) Induction of donor-specific unresponsiveness by intrathymic islet transplantation Science 249,1293-1295[Abstract/Free Full Text]
  36. Bonomo, A., Kehn, P. J., Shevach, E. M. (1995) Post-thymectomy autoimmunity: abnormal T-cell homeostasis Immunol. Today 16,61-67[Medline]
  37. Salaun, J., Banderia, A., Khazaal, I., Calman, F., Coltey, M., Coutinho, A., Le Douarin, N. M. (1998) Thymus requirement and antigen dependency in the infectious tolerance pathway in transplant recipients J. Am. Assoc. Immunol. 160,5765-5772
  38. Onodera, J., Volk, H. D., Ritter, T., Weglinski, (1998) Thymus requirement and antigen dependency in the infectious tolerance pathway in transplant recipients J. Am. Assoc. Immunol. 160,5765-5772
  39. Khoury, S. J., Gallon, L., Chen, W., Betres, K., Russell, M. E., Hancock, W. W., Carpenter, C. B., Sayegh, M. H., Weiner, H. L. (1995) Mechanisms of acquired thymic tolerance in experimental autoimmune encephalomyelitis: thymic dendritic-enriched cells induce specific peripheral T cell unresponsiveness in vivo J. Exp. Med. 182,357-366[Abstract/Free Full Text]
  40. Durkin, H. G., Chice, S., Goldschneider, I. (1984) Origin and fate of antigen-specific suppressor T cells Ann. N. Y. Acad. Sci. 435,258-259
  41. Hayamizu, K., Huie, P., Sibley, R. K., Strober, S. (1998) Monocyte-derived dendritic cell precursors facilitate tolerance to heart allografts after total lymphoid irradiation Transplantation 66,1285-1291[Medline]
  42. Oluwole, S. F., Jin, M.-X., Chowdhury, N. C., Englestad, K., Ohajekwe, A., T. A, J. (1995) Induction of peripheral tolerance by intrathymic inoculation of soluble alloantigens: evidence for the role of host antigen-presenting cells and suppressor cell mechanism Cell. Immunol. 162,33-41[Medline]
  43. Fu, F., Li, Y., Qian, S., Lu, L., Chambers, F., Starzl, T.E., Fung, J. J., Thomson, A. W. (1996) Costimulatory molecule-deficient dendritic cell progenitors (MHC-class II+, CD80dim, CD86-) prolong cardiac allograft survival in nonimmunosuppressed recipients Transplantation 62,659-665[Medline]
  44. Lu, L., McCaslin, D., Starzl, T. E., Thomson, A. W. (1995) Bone marrow-derived dendritic cell progenitors (NLDC 145+, MHC class II+, B7-1dim, B7-2-) induce alloantigen-specific hyporesponsiveness in murine T lymphocytes Transplantation 60,1539-1545[Medline]
  45. Williamson, E., Westrich, G. M., Viney, J. L. (1999) Modulating dendritic cells to optimize mucosal immunization protocols J. Immunol. 163,3668-3675[Abstract/Free Full Text]
  46. Erwig, L.-P., Kluth, D. C., Walsh, G. M., Rees, A. J. (1998) Initial cytokine exposure determines function of macrophages and renders them unresponsive to other cytokines J. Immunol. 161,1983-1988[Abstract/Free Full Text]
  47. Liu, L., Rich, B. E., Inobe, J., Chen, W., Weiner, H. L. (1998) Induction of Th2 cell differentiation in the primary immune response: dendritic cells isolated from adherent cell culture treated with IL-10 prime native CD4+ T cells to secrete IL-4 Int. Immunol. 10,1017-1026[Abstract/Free Full Text]
  48. Rastellini, C., Lu, L., Ricordi, C., Starzl, T. E., Rao, A. S., Thomson, A. W. (1995) Granulocyte/macrophage colony-stimulating factor-stimulated hepatic dendritic cell progenitors prolong pancreatic islet allograft survival Transplantation 60,1366-1370[Medline]



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