* Department of Pathology and
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
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,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
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We have recently demonstrated that an intact thymus is required for the
induction of ACAID in AC-injected mice and that ,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.
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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.
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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.
 View larger version (42K): [in a new window] |
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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 View larger version (33K): [in a new window] |
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.
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,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.
 View larger version (44K): [in a new window] |
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.
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 View larger version (37K): [in a new window] |
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.
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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.
 View larger version (24K): [in a new window] |
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.
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 View larger version (26K): [in a new window] |
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.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 ,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
.
 View larger version (15K): [in a new window] |
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 ,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).
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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.
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.
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