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*
Dumont-University of California at Los Angeles Transplant Center, Department of Surgery, University of California School of Medicine, Los Angeles, CA 90095;
Institute of Medical Biochemistry, University of Rostock, Rostock, Germany; and
Institute of Medical Immunology, Humboldt University, Berlin, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously reported that 1) treatment with RIB-5/2, a CD4-nondepleting mAb, produces indefinite survival of LBNF1 cardiac allografts in presensitized Lewis (LEW) rat recipients (4); 2) donor-specific and organ nonspecific tolerance can be then transferred by spleen cells into new cohorts of test graft recipients (4); 3) regulatory CD4+ T cells are instrumental in this model, as their selective depletion prevents the acquisition of tolerance (4); 4) thymus-dependent regulatory CD4+ T cell-mediated effective memory and suppression depend on the presence of donor Ag rather than "graft adaptation" (7); and 5) selective up-regulation of IL-4 at the graft site is useful for tolerance maintenance in test rat recipients conditioned with regulatory T cells (8). Hence, both local type 2 cytokine expression pattern in the graft itself and circulating regulatory T cells influence allograft outcome in the infectious tolerance pathway (9).
Our current knowledge of the infectious tolerance mechanisms derives primarily from in vivo experiments in animal transplantation models. Indeed, tolerogenic lymphocytes in such allograft recipients have not been characterized in vitro, which hampers our appreciation of the cellular and molecular mechanisms underlying this dominant immune regulation pathway. In this study, we attempted to establish an in vitro cell culture system that would recapitulate some cardinal in vivo features of the infectious tolerance pathway. Our results demonstrate: 1) selective compartmentalization of tolerogenic IL-2-responsive T lymphocytes in the spleen of long term allograft recipients; 2) vigorous in vivo proliferation of T cells from tolerant hosts in allogeneic donors; 3) the ability of splenocytes from tolerant hosts to suppress proliferation of naive splenocytes in the coculture assay, an ultimate test for a dominant immune regulation; and 4) the CD45RC+ subset in tolerogenic T cells hyporesponsive to alloantigen and able to impose the suppressive function upon normal T cells. To the best of our knowledge, these studies are the first to provide direct evidence that a nondeletional, anergy-like regulatory mechanism(s) might operate via a discrete T cell subset in the infectious tolerance pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inbred male adult rats, weighing 200–250 g, were used (Harlan Sprague-Dawley, Indianapolis, IN). LEW (RT1l) rats served as recipients of cardiac allografts from (LEW x BN)F1 (LBNF1) hybrids. Brown-Norway (BN; RT1n) rats were used as skin donors, and Wistar-Furth (WF; RT1u) served as heart/cell donors for specificity studies. Full thickness skin was sutured bilaterally to appropriate defects in the chest of prospective recipients. Hearts were transplanted to the abdominal great vessels by standard microvascular techniques. Graft function was monitored daily by palpation, and rejection was defined as the day of cessation of heartbeat.
Allograft model
LEW rats were sensitized with BN skin grafts (day -7), followed 1 wk later by transplantation of LBNF1 hearts (day 0). These cardiac allografts are rejected in an accelerated manner in <36 h (acute rejection occurs in 7–8 days) (10). Animals were treated with CD4 nondepleting (RIB-5/2) mAb (5 mg/rat i.v. on days -7 (day of skin graft), -4, -1, and 0 (day of heart graft) and on days 1, 4, 7, 10, 14, and 21 thereafter). This regimen induces >100-day acceptance of cardiac allografts, with concomitant cardinal features of the infectious tolerance pathway (4, 7, 8, 9).
MLR in vitro and in vivo
Rat splenocytes or lymph node (LN)3 cells were prepared as single-cell suspensions. RBC were lysed with RBC lysis buffer (Sigma, St. Louis, MO). After washing twice with RPMI 1640 medium and 1% FBS, cells were suspended in culture medium (RPMI 1640 supplemented with 20 mM HEPES, 10 mM sodium pyruvate, 2 mM L-glutamine, 50 nM 2-ME, 1x MEM-nonessential amino acid solution, 1x MEM-vitamin solution, 1x antibiotic/antimycotic solution, and 10% FBS) at 5 x 106/ml. One hundred microliters of LEW lymphocytes were added to a U-bottom 96-well plate (Corning Glass, Corning, NY), mixed with the same volume of gamma-irradiated (2000 rad) allogeneic donor-type (BN/LBNF1) or third-party (WF) splenocytes in the presence of 2 mM NG-monomethyl-L-arginine, an inhibitor of the macrophage form of NO synthase. There were four replicates for each reaction combination. Con A (2 µg/ml) was used as a positive control. [3H]TdR (1 µCi) was added to each well in the last 16–18 h of culture. Labeled cells were harvested onto filtermats (Skatron, Sterling, VA) with a Skatron 12-well semiautomatic cell harvester. The cpm of the filter membrane were measured in scintillation liquid (Cytoscint; ICN Biomedical, Costa Mesa, CA) on an LS 6000IC (Beckman Coulter, Palo Alto, CA).
In in vivo MLR, LEW rat lymphocytes were labeled with CFSE (Molecular
Probes, Eugene, OR) at 4 mM in PBS for 15 min at 37°C. The
unconjugated CFSE was eliminated by washing the cells with FBS
(20%)-supplemented RPMI medium. The labeled cells were resuspended in
PBS at 5 x 107/ml, and 2 ml of these cells
(1 x 108) were injected into
gamma-irradiated (1000 rad) BN (or LEW as negative controls) rats via
the tail vein. On day 3, spleens were harvested, and splenocytes were
stained with anti-rat TCR 
-R-PE (R73), CD4-CyChrome (OX-35,
BD PharMingen, San Diego, CA). Topro 3 (1 nM; Molecular Probes) was
added as a viable dye. Four-color flow cytometry was performed on a
FACSCalibur dual-laser cytometer (BD Biosciences, Mountain View, CA).
Topro 3-negative (viable) cells in the lymphocyte gate stained
positively for TCR and CD4 were analyzed for CFSE
intensities.
Adoptive cell transfer studies
To determine the presence of regulatory T cells, we performed
adoptive transfer studies in which 100 x
106 erythrocyte-free spleen or LN cells were
administered i.v. to lightly gamma-irradiated (450 rad) syngeneic
secondary recipients. These were challenged 24 h later with
donor-specific test cardiac allografts. We have used this in vivo
adoptive cell transfer system previously (4, 7, 8, 9).
Indeed, gamma-irradiation does not affect host alloreactivity per se,
as the majority of these recipients reject their transplants in a
normal acute fashion (8–10 days), elicit a brisk (
4-day) rejection
after transfer of splenocytes from sensitized rejecting controls, yet
maintain test grafts indefinitely after infusion of spleen cells from
CD4 mAb-pretreated long term tolerant hosts. The cells successfully
engraft after adoptive transfer, and their immune potential may be
accurately determined in gamma-irradiated "test-tube" rats.
Isolation of T cell subsets
Single-cell spleen cell suspensions (1–2 x 108) were enriched for T cells with a nylon wool column (10 ml; PolyScience, Warrington, PA). T cells were then incubated with CD45RC mAb (OX-22, BD PharMingen) at 4 C for 30 min (2 x 107 cells/5 µg Abs). After washing, Dynal beads coated with anti-mouse IgG (CELLection Pan Mouse IgG Kit, Dynal, Lake Success, NY) were added to the Ab-coated T cells to separate negatively selected CD45RC+ and CD45RC- cells, according to the manufacturer’s manual. Cell subsets were resuspended at 1–2 x 107/ml, and cells (100 µl/well) were used for MLRs. In mixing cultures, 1 x 106 CD45RC+ cells or 2 x 106 CD45RC- cells were mixed with 5 x 106 T cells from naive or rejecting recipients.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lymphocytes from groups of age-matched untreated LEW hosts
(rejected BN skin and LBNF1 cardiac grafts), and
long term (>100 days) tolerant CD4 mAb-treated cardiac graft
recipients were analyzed for in vitro alloreactivity in the MLR assay.
As shown in Fig. 1
A,
splenocytes, but not LN cells, from tolerant hosts had significantly
lower proliferative responses against donor-type alloantigen; the
magnitude of cell proliferation within the tolerant splenocyte pool was
depressed to 15–25% of that in rejecting controls. Polyclonal
activation with Con A stimulated proliferation of all T cell groups,
indicating no general defects in the ability of proliferation in
tolerant splenic T cells. FACS staining of CFSE-labeled lymphocytes
after 4-day culture showed the CD4+ T cells to be
the dominant cell type in the alloresponsive population and confirmed
diminished frequency of tolerant cells that proliferated after
alloantigen stimulation (Fig. 1B). Addition of Con A
restored CD4+ T cell proliferation in vitro.
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We then performed adoptive transfer experiments to correlate T cell hyporesponsiveness in vitro with their in vivo ability to suppress allograft rejection. Groups of lightly irradiated (450 rad) secondary syngeneic recipients were infused with either splenocytes or LN cells (100 x 106) from tolerant donors (>100 days post-transplant), followed 1 day later by cardiac engraftment. Indeed, in agreement with our previous findings (4), all test cardiac allografts survived long term (>100 days) in secondary recipients, which were injected with tolerant splenocytes (n = 6; data not shown). In contrast, test cardiac allograft survival of 8.4 ± 2.3 days (mean ± SD; n = 7) after transfer of LN cells from tolerant hosts was not different from those recorded in irradiated, otherwise unmodified recipients (10.3 ± 7.3 days; n = 12) or in those given normal naive cells (6.8 ± 0.5 days; n = 4). Collectively, these results document the compartmentalization of tolerogenic T lymphocytes in the infectious tolerance pathway with spleen, but not LN, cells showing hyporesponsiveness to alloantigen stimulation in vitro as well as operational activity in the in vivo adoptive transfer system. Therefore, to explore putative immune regulation mechanisms leading to transplantation tolerance, we focused on spleen cells from long term cardiac allograft recipients.
In vivo MLR reveals the existence of alloreactive CD4+ T cells in tolerant splenocytes
A number of interlocked immune mechanisms may contribute to
hyporesponsiveness in vitro and in vivo. The deletion of alloreactive T
cells irreversibly decreases clonal size in the lymphocyte population.
In addition, regulatory T cells, induced under the cover of
CD4-targeted therapy, may act on otherwise alloreactive T cells to
prevent their activation within a normal lymphocyte pool. In the latter
case, close cell-cell contact may be required (11). In
vivo MLR provides an ideal setting to differentiate between deletional
and nondeletional mechanisms. Hence, CFSE-labeled splenocytes from
tolerant LEW recipients of LBNF1 heart grafts
(>100 days post-transplant) were injected i.v. into gamma-irradiated
(1000 rad) BN rats to determine their alloreactivity in vivo. The
results presented in Fig. 2 are in
agreement with robust proliferation of transferred tolerant cells,
similar to that of rejecting controls. This response was allospecific,
as no significant proliferation was detected with cells injected into
gamma-irradiated syngeneic (LEW) rats. In agreement with others
(12), CD4+ T cells were the major
responsive population in in vivo MLR. These results indicate that
alloreactive T cells do exist in the tolerogenic splenocyte pool, and
that regulatory T cells may impose immunosuppression in vitro via a
close cell-cell contact. However, in the in vivo setting with
regulatory and other T cells dispersed, activation and proliferation of
alloreactive T cells may readily occur.
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To examine the role of IL-2 in the hyporesponsiveness of
tolerogenic splenocytes against alloantigen, exogenous IL-2 was added
to the in vitro MLR. As shown in Fig. 3, with increasing doses of IL-2 the magnitude of proliferation was
boosted in tolerogenic splenocyte cultures. However, the dose of IL-2
required to restore normal proliferation in vitro was relatively high.
Indeed, IL-2 at 10 U/ml, a dose known to recover anergic T cells
(13), increased the proliferation level only to 33% of
that in the control. The allospecific proliferation indexes did not
change significantly, as inclusion of IL-2 boosted syngeneic cell
proliferation as well. Thus, two possibilities can be envisioned: 1)
tolerogenic splenocytes may have contained an anergic T cell component;
or 2) IL-2 could abrogate the regulatory T cell-driven suppression.
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One of the cardinal features of regulatory T cells in vivo is
their ability to prevent other alloreactive T cells from rejecting the
grafts (3, 4). To mimic such a regulatory function in
vitro, we have established a coculture system in which equal numbers of
splenocytes from different sources were mixed (1:1 ratio), and the
resulting total proliferation was analyzed. Fig. 4 depicts results of a typical mixing
experiment in which tolerogenic and normal splenocytes were added in
concert to in vitro MLR. Interestingly, tolerogenic splenocytes not
only lost alloreactive responses against alloantigen, but also
suppressed the proliferation of normal splenocytes. Indeed, a
half-million tolerogenic splenocytes mixed with the same number of
normal splenocytes at the start of a 4-day culture reduced the
proliferation of normal splenocytes to 10% compared with that of
cultured cells with alloantigen alone. Thus, spleen cells in animals
tolerized in an infectious manner can disable naive lymphocytes so that
they fail to proliferate in vitro.
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We have previously shown that a population of
CD4+ T cells was responsible for immune
suppression in vivo, as evidenced by prevention of test allograft
rejection in an adoptive transfer system (4). To further
dissect the immunoregulatory T cell network, we separated T lymphocytes
based on their CD45RC levels. As shown in Fig. 5, if CD25 staining was applied to CD45RC
subsets, most CD25+ cells were detected in the
CD45RC- T cell fraction. Both
CD45RC+ and CD45RC- T
cells from rejecting recipients responded vigorously to alloantigen in
in vitro MLR (Fig. 6). In contrast,
although unseparated T cells from tolerant hosts revealed a lower
proliferative response to donor Ag, the CD45RC-
subset showed a relatively normal response. The hyporesponsiveness of
tolerant T cells was restricted selectively to the
CD45RC+ subset.
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Consistent with our results from one-way MLR,
CD45RC+, but not CR45RC-,
T cells from tolerant recipients imposed suppressive function and
significantly (p < 0.007) inhibited the
proliferation of normal T cells against alloantigen in the coculture
assay (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The divergent ability of lymphocytes from spleen and LN to affect in vitro and in vivo alloimmune responses illustrates a previously overlooked aspect, i.e., the compartmentalization of the putative regulatory T cells in long term tolerant graft recipients. Indeed, only splenocytes from CD4 mAb-treated tolerant rats had a markedly reduced allo-proliferative response in MLR. Furthermore, tolerant spleen, but not LN, cells were capable of suppressing the proliferation of naive splenocytes in the coculture assay. Others have shown that LN cells have lower proliferative responses against Con A stimulation compared with splenocytes, possibly due to the relative lack of accessory cells (14). Consistent with our present in vitro data, spleen, but not LN, cells adoptively transferred transplantation tolerance into new cohorts of syngeneic test animals. Thus, it is plausible that regulatory T cells preferentially accumulate in the spleen, or the frequency of regulatory T cells in the spleen is much higher compared with that in the LN compartment. Recent studies have also shown that regulatory T cells may express a memory-type phenotype (15, 16). Perhaps memory T cells fail to sequester in LN because they lack L-selectin Ags on their surface (17). Indeed, we have consistently observed much lower frequency of CD3+CD4+CD45RClow T cells in LN compared with the spleen (14 vs 38%) of tolerant rat recipients (Y. Zhai and J. W. Kupiec-Weglinski, unpublished observations). Alternatively, the development of regulatory T cells, which requires the direct alloantigen contact may occur preferentially in recipient’s spleen. Moreover, preliminary data show a very early accumulation of regulatory T cells in the graft itself, i.e., at the site of direct Ag contact (B. Sawitzki and H.-D. Volk, manuscript in preparation). Collectively, although we favor the idea that regulatory T cells recognize Ag through direct allo-presentation, we cannot exclude a possible role of indirect presentation in our experimental system. Indeed, the latter has been recently suggested to play a role in the acquisition of tolerance in a mouse skin transplant model (18).
It was initially surprising to find out that CFSE-labeled tolerogenic splenocytes proliferated vigorously in vivo following infusion into allogeneic test rats, considering their relatively low allogeneic responsiveness in vitro. However, because alloreactive T cells were not deleted in the tolerogenic splenocyte pool in our model, the in vivo MLR setting may have disrupted the conditions required for active immune regulation. As shown in some autoimmune disease models, close cell-cell contact may be required for regulatory T cells to exert immunosuppressive effects on other T cells (11). It is doubtful that regulatory T cells and other alloreactive T cells interact closely with APCs in the alloantigen-loaded environment of allogeneic recipients. The situation is different in adoptively transferred test allograft recipients, in which regulatory T cells are able to prevent T cell activation, because the site of Ag contact is restricted to the graft and the draining lymph nodes, allowing a more direct cell-cell contact similar to that occurring in in vitro cultures. Alternatively, in vivo milieu (e.g., high IL-2 levels induced by gamma irradiation) may break the anergy state of alloreactive T cells or abrogate suppression of alloreactive lymphocytes by regulatory T cells (10). Consistent with the latter hypothesis, a high dose of exogenous IL-2 successfully restored allo-proliferative in vitro response of tolerogenic splenocytes in this study.
The recapitulation of in vivo tolerogenic effects of splenocytes from
CD4 mAb-pretreated recipients in in vitro cell culture system provides
us with a unique opportunity to dissect immune mechanisms at work in
the infectious tolerance pathway. The isolation of a T cell subset that
imposes suppression on other T cells is also consistent with our in
vivo MLR studies, which showed persistence of alloreactive cells in the
tolerant splenic T cell pool. Two major regulatory pathways in
autoimmune disease models involve cytokines and cell-cell contact
(19). Rat regulatory CD4+ T cells
were found to be of CD45RClow phenotype and to
produce IL-2 and IL-4, but not IFN-
, upon in vitro stimulation
(20). Moreover, IL-4 and TGF- are critical in
preventing autoimmunity, as neutralization of either of the two
cytokines abrogated the protective effects of normal syngeneic
CD4+ T cells in the adoptive transfer system
(21). Our finding that the tolerant
CD45RC+ population contained regulatory T cells,
which were both hyporesponsive to alloantigen stimulation and able to
suppress proliferation of naive T cells in vitro, indicates that cells
involved in the acquisition of transplantation tolerance might
represent a different regulatory T cell lineage compared with those in
autoimmunity. A subset of memory CD4+ T cells,
identified by the naive phenotype of CD45RC+, has
been shown to derive from CD45RC- memory cells
in the absence of stimulating alloantigen and to be relatively
long-lived compared with CD45RC- memory T cells
(22). The tolerant splenocytes in our studies were
harvested from recipients bearing long term (>100 days) cardiac
allografts. As donor-type APCs (direct allo-recognition) for CD4 T
cells at this late time point may have been diminished,
CD45RC+ regulatory T cells in this study may well
represent CD45RC- "revertants."
Although inhibition of the normal T cell response by tolerant CD45RC+ in the coculture assays was not that dramatic, it was statistically significant (p < 0.007). These cells went through extensive manipulation in vitro, and the culture conditions may not have been optimal to exert their immunosuppressive functions. Therefore, it is reasonable to suggest that under optimal conditions and without much in vitro stress, the tolerant CD45RC+ T cells will be much more effective in suppressing other cells in vivo. Indeed, future adoptive transfer experiments should determine whether the CD45RC+ T cell subset exerts similar allo-hyporesponsive and suppressive properties in vivo. As for the CD25 phenotype, we have observed that most of the CD25+ T cells resided within the CD45RC- subset in both tolerant and rejecting recipients’ spleens. Because the efficacy of our Dynal bead-assisted separation of CD25+ T cells was very low, we were unable to set up sufficient numbers of CD25+ cells in MLRs. However, based on the CD25 staining profile and CD45RC subset results, we suspect that CD25+ cells in tolerant recipients will be characterized by relatively normal proliferative responses.
We have detected increased production of IL-4 and IL-10 in tolerant splenocytes even after polyclonal T cell activation (Y. Zhaiand J. W. Kupiec-Weglinski, unpublished observations). Such a Th2-type deviation was associated with the tolerant state and restricted to a small population of CD4+ T cells. This correlates with the results of our adoptive transfer studies in which infusion of tolerogenic spleen cells led to selective up-regulation of Th2-type cytokines at the graft site of secondary test recipients (8), whereas depletion of CD4+ cells in the transferred inoculum prevented such an up-regulation and abolished the infectious tolerance pathway (4).
In addition to soluble mediators, cell-cell contact may be critical in
alloimmune regulation (23). As shown by several recent
studies, anergic T cells can act as suppressor cells both in vitro and
in vivo, possibly via a mechanism of competition for the APC
surface and locally produced IL-2 (13, 24, 25). In mouse
autoimmune disease models, anergic CD4+
regulatory T cells express IL-2R -chain and fail to proliferate upon
TCR stimulation, yet they effectively suppress the proliferation of
naive T cells (11). Exogenous IL-2 or anti-CD28 mAb
restore the proliferation of
CD25+CD4+ T cells by
anti-CD3 stimulation and abolish suppression in a
CD25- and CD25+ T cell
mixture (11, 26).
In summary, we have established an in vitro experimental system to analyze the function of regulatory T cells from an in vivo model of infectious transplantation tolerance in rats. Our results are the first to provide direct evidence for nondeletional, anergy-like active regulatory mechanisms that operate in vitro via the CD45RC+ subset in the splenic, but not LN, compartment of tolerant hosts. Future in vitro studies should identify distinctive CD4+ T cell subsets (CD25, CD62 ligand) that may give rise to the alloimmune regulatory T cells and determine whether close cell-cell contact and APCs, on the one hand, and type 2 cytokines, on the other, are indeed required for the acquisition of infectious tolerance in transplant recipients.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jerzy W. Kupiec-Weglinski, Dumont-University of California at Los Angeles Transplant Center, Room 77-120 CHS, 10833 Le Conte Avenue, Los Angeles, CA 90095. E-mail address: jkupiec{at}mednet.ucla.edu
3 Abbreviation used in this paper: LN, lymph node.
Received for publication May 31, 2001. Accepted for publication August 9, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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