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,
*
Program in Immunology and Virology, and Departments of
Medicine,
Surgery, and
Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605
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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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Combined therapy consisting of anti-CD154 mAb plus a single donor-specific transfusion (DST)5 in mice is even more effective than anti-CD154 mAb monotherapy in prolonging the survival of islet, skin, and heart allografts (5, 6, 15, 16, 17, 18, 19). The mechanism by which DST enhances graft survival appears in part to involve the deletion of recipient CD8+ alloreactive T cells (15). Combined therapy consisting of anti-CD154 mAb plus CTLA4-Ig also leads to deletion of alloreactive CD8+ T cells and prolonged allograft survival (20, 21, 22).
Most skin allografts placed on mice treated with any of these protocols are eventually rejected (23) unless recipients are thymectomized (18). Skin allografts can actually survive indefinitely on thymectomized mice treated with DST and anti-CD154 mAb (18, 24). These observations have led us to hypothesize that allograft rejection in tolerized mice is due to the emergence of alloreactive thymic emigrants in a milieu in which declining levels of anti-CD154 mAb preclude the blockade of costimulation (5, 18). In support of this hypothesis, we (25) and others (26, 27) have shown that hemopoietic stem cell reconstitution of mice with successful intact allografts will lead to the rejection of these grafts in the absence of surgical trauma or other forms of activation, but direct evidence that this is due to newly developed T cells is lacking.
To test this hypothesis more directly and to analyze the underlying mechanisms in detail, we established a new analytical system based on allo-TCR transgenic hemopoietic chimeric graft recipients. Chimeras were normal CBA (H-2k) mice that were irradiated and given small numbers of syngeneic TCR-transgenic KB5 bone marrow cells. These mice subsequently circulated a self-renewing trace population of anti-H-2b alloreactive CD8+ T cells that matured in a normal microenvironment. With this system, we studied the immune response to H-2b allografts in tolerized mice. We documented that alloreactive CD8+ T cells were deleted during tolerance induction, but slowly returned toward pretreatment levels in euthymic mice. Skin allograft rejection in this system occurred in the context of increasing numbers of alloreactive CD8+ cells.
Using this new model system, we also tested the hypotheses that 1) declining concentrations of anti-CD154 mAb in the circulation, 2) deletion of CD4+ regulatory cells, and 3) activation of alloreactive CD8+ cells would correlate with eventual graft rejection. Each of these hypotheses was confirmed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CBA/JCr (H-2k), C57BL/6 (H-2b), and BALB/c (H-2d) mice were obtained from the National Cancer Institute (Frederick, MD). (KB5 x CBA/JCr)F1 TCR-transgenic mice were obtained from a colony maintained in our facility (28). The founders were the generous gift of Dr. J. Iacomini (Harvard Medical School, Boston, MA), who obtained the mouse from the original developer, Dr. A. Mellor (Medical College of Georgia, Augusta, GA). The TCR transgene is expressed in CBA (H-2k) mice by CD8+ cells, and the transgenic TCR has specificity for native H-2-Kb (29).
All animals were certified to be free of Sendai virus, pneumonia virus of mice, murine hepatitis virus, minute virus of mice, ectromelia, lactate dehydrogenase-elevating virus, mouse poliovirus (GD7), Reo-3 virus, mouse adenovirus, lymphocytic choriomeningitis virus, polyoma, Mycoplasma pulmonis, and encephalitozoon cuniculi. All animals were housed in microisolator cages, given ad libitum access to autoclaved food, and maintained in accordance with recommendations in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, National Academy of Sciences, 1996) and the guidelines of the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School.
Generation of KB5 TCR-transgenic hemopoietic CBA chimeras
To examine the fate of both mature and developing alloreactive CD8+ T cells in a normal microenvironment, we used TCR-transgenic hemopoietic chimeras (30) generated by injecting small numbers of bone marrow cells from KB5-transgenic donors into sublethally irradiated syngeneic CBA-nontransgenic hosts. We refer to these as synchimeric mice. Marrow donors were male and female (KB5 x CBA/JCr)F1 mice, 8–12 wk of age. Donor femurs and tibias were flushed with RPMI 1640 medium using a syringe with a 24-gauge needle. Recovered cells were filtered through sterile nylon mesh (70 µm; BD Biosciences, Franklin Lakes, NJ), counted, and resuspended in RPMI 1640. Recipients were male CBA/JCr mice 4–7 wk of age. Recipient mice were treated with whole body gamma irradiation using a 137Cs source (Gammacell 40; Atomic Energy of Canada, Ottawa, Ontario, Canada). They were then given a single i.v. injection of KB5 bone marrow cells in a volume of 0.5–1 ml via the lateral tail vein within 2–5 h of irradiation. The transgenic T cells that develop from KB5 bone marrow express an anti-H-2-Kb-specific TCR that is recognized by the clonotypic mAb DES (29). In preliminary dosing experiments, it was determined that 2 Gy radiation and 0.5 x 106 KB5 bone marrow cells were optimal for the generation of hemopoietic chimeras circulating DES+CD8+ T cells (see Results), and these parameters were used in all experiments reported in this work.
Flow microfluorometry
A mouse hybridoma cell line secreting the KB5-specific
clonotypic DES Ab (29) was a gift from Dr. J. Iacomini
(Harvard Medical School). FITC-conjugated anti-mouse IgG2a
developing reagent for DES (clone R19-15), PerCept-conjugated and
CyChrome-conjugated anti-mouse CD8
mAbs (both clone 53-6.7), and
PE-conjugated anti-mouse mAb directed against the activation marker
CD62L (Mel-14) were obtained from BD PharMingen (San Diego, CA).
Isotype control mAbs including the PE-conjugated rat IgG2b 
(clone
A95-1) for CD62L, rat PerCept-conjugated and CyChrome-conjugated IgG2a
(clone R35-95) for CD8, and mouse IgG2a
anti-trinitrophenol (clone G155-178) for DES were purchased
from BD PharMingen.
Two- and three-color flow microfluorometry analyses of lymph node and spleen cells were performed, as previously described (15). Briefly, 1 x 106 viable cells were reacted with anti-DES Ab for 20 min at 4°C. Cells were then washed and reacted with FITC-conjugated anti-mouse IgG2a mAb (to develop the DES Ab) plus a mixture of conjugated mAbs for 20 min. Whole blood was processed using FACS lysing solution (BD Biosciences) in accordance with the protocol supplied by the manufacturer. Labeled cells were washed, fixed with 1% paraformaldehyde-PBS, and analyzed using a FACScan instrument (BD Biosciences). Lymphoid cells were gated according to their light-scattering properties, and 30–50 x 103 events were acquired for each analysis.
Transplantation procedures
Male CBA/JCr and KB5 synchimeric CBA/JCr mice were treated with DST and anti-CD154 mAb and transplanted with skin allografts, as described elsewhere (15, 18, 19). Briefly, the DST consisted of 107 C57BL/6 splenocytes from 5- to 10-wk-old female donors injected i.v. in a volume of 0.5 ml. DST was given on day -7 relative to skin transplantation. MR1 hamster anti-mouse CD154 mAb was produced as ascites in SCID mice and purified by affinity chromatography (31, 32). Ab concentration was determined by measurement of OD and confirmed by ELISA (see below). The concentration of contaminating endotoxin was determined commercially (Charles River Endosafe, Charleston, SC) and was uniformly <10 U/mg mAb (31). Mice were injected i.p. with anti-CD154 mAb (0.5 mg/dose) on days -7, -4, 0, and +4 relative to skin transplantation. Full-thickness skin grafts 1–2 cm in diameter were obtained from the flanks of donor mice and transplanted onto the dorsal flanks of recipients, as described previously (18). Graft rejection was defined as the first day on which the entire graft was necrotic (18, 19).
The KB5 synchimeras received skin allografts when 11–17 wk of age, 7–12 wk after irradiation and bone marrow injection, to establish hemopoietic chimeras. Wild-type CBA/JCr mice received skin grafts when 6–14 wk of age.
Concentration of hamster anti-mouse CD154 mAb
To measure the concentration of anti-CD154 mAb in serum and in preparations of purified mAb, we developed an ELISA specific for hamster IgG. Briefly, high protein-binding affinity 96-well ELISA plates (Costar, Cambridge, MA) were incubated overnight at 4°C with affinity-purified goat anti-hamster IgG Ab (Boehringer Mannheim, Indianapolis, IN) in 0.1 M Na2HPO4 (pH 9), washed with PBS, and blocked with 0.1% BSA in PBS. Plates were washed with PBS containing 0.05% Tween 20 (PBS-Tween). Samples and standards prepared in our laboratory were diluted as required in PBS-Tween, incubated overnight at 4°C, washed with PBS-Tween, and incubated with excess biotinylated goat anti-hamster IgG Ab (Vector Laboratories, Burlingame, CA), avidin-HRP conjugate (Vector Laboratories), and the peroxidase substrate o-phenylene diamine (OPD; Sigma, St. Louis, MO). Plates were washed between incubations with PBS-Tween. Before the addition of OPD, the plates were washed in PBS alone. OPD (final concentration 1 mg/ml) and H2O2 (0.003%) were added to sterile substrate buffer (0.05 M Na2HPO4, 0.025 M sodium citrate, pH 5) just before use. The reaction was stopped by addition of 3 M HCl, and absorbance was read at 490 nm using a plate-reading spectrophotometer (Bio-Rad model 3550; Bio-Rad, Hercules, CA).
Concentration of mouse anti-hamster Ig
To measure the serum concentration of mouse anti-hamster Ig Ab produced in response to the injection of anti-CD154 mAb, 96-well ELISA plates (Costar) were coated with 50 µl of a 5 µg/ml solution of MR1 Ab in PBS. Plates were washed and blocked as described above. A standard mouse serum was produced by immunization of a pool of C57BL/6 mice with hamster Ig (20 µg; Jackson ImmunoResearch Laboratories, West Grove, PA) in CFA. After 2 wk, the animals received an i.v. boost of 50 µg of hamster Ig. Serum was recovered 7 days later, divided into aliquots, and stored at -70°C until used. This standard positive control serum was assigned an arbitrary value of 100,000 U/ml anti-hamster IgG-binding activity. MR1 hamster Ig-coated plates were incubated with dilutions of the standard serum and serum from experimental animals. After overnight incubation, bound mouse Ab was detected with affinity-purified biotinylated goat anti-mouse IgG (Vector Laboratories), avidin-HRP, and OPD, as described above.
Hemagglutination titers in mice immunized with SRBC
Anti-CD154 mAb prevents T-dependent Ab responses both in vivo and in vitro (1, 2, 33). To determine the serum concentration of anti-CD154 mAb required to achieve this effect, we used an SRBC hemagglutination assay. C57BL/6 mice were given a single i.v. injection of 0.2 ml of 1% SRBC (Colorado Serum, Denver, CO). Immediately thereafter, the mice were randomized into six groups. One group received no further treatment; the others were treated with a single injection of anti-CD154 mAb at five dosage levels that ranged from 0.016 to 0.5 mg. Serum samples were collected from control and experimental mice immediately before and 2, 7, and 14 days after treatment. SRBC hemagglutination titers were assayed as described previously (34). Briefly, serial 2-fold dilutions of heat-inactivated mouse sera were placed in 96-well round-bottom plates (50 µl/well). Each assay included dilutions of serum obtained from control mice that received SRBC only and control wells containing PBS alone. To each well was added 50 µl of 1% SRBC in PBS (washed packed cells/total volume). The plates were covered and the cells were allowed to settle undisturbed overnight at room temperature. The SRBC hemagglutination titer was defined as the highest dilution that produced visible agglutination.
Statistics
Average duration of graft survival is presented as the median. Graft survival among groups was compared using the method of Kaplan and Meier (35). The equality of allograft survival distributions for animals in different treatment groups was tested using the log rank statistic (35). The p values <0.05 were considered to be statistically significant. Comparisons of two means used unpaired t tests (36). Regression curves were fitted using SigmaPlot software (version 6.0; SPSS, Chicago, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To generate KB5 TCR-transgenic hemopoietic chimeras, we first
performed preliminary experiments to determine appropriate doses of
radiation and bone marrow. These revealed that the combination of 2 Gy
radiation and 0.5 x 106 KB5 bone marrow
cells generated chimeras that circulated 2–8%
DES+CD8+-transgenic T cells
in the peripheral blood within 6–8 wk (data not shown). These
parameters were used in all subsequent experiments. Overall, 
90% of
mice treated when 4–7 wk of age became chimeric, defined as the
presence of 
2%
DES+CD8+-transgenic T cells
8 wk after bone marrow injection.
We then determined the stability of the chimeric state in CBA
recipients that were not otherwise manipulated. As shown in Fig. 1
A, the percentage of
DES+CD8+-transgenic T cells
was similar in all mice and remained quite stable between 50 and 197
days after bone marrow injection. The data were fitted by linear
regression, and the slope of the regression line is statistically
indistinguishable from 0, indicating that the average number of
circulating transgenic T cells remained constant. The overall
percentage of
DES+CD8+-transgenic T cells
during this interval was 7.4 ± 1.4% (n = 56
measurements in five mice) and ranged from 6.6 ± 0.9 to 8.5
± 1 for individual mice at different time points. The minimum
percentage in any animal at any time point was 4.4% and the maximum
was 9.6%.
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We first demonstrated that the KB5 TCR-transgenic hemopoietic CBA
chimeras respond to treatment with DST plus anti-CD154 mAb by
reducing existing DES+CD8+
alloreactive T cells in peripheral blood. Chimeras received a single
transfusion of C57BL/6 spleen cells, a 2-wk course of anti-CD154
mAb, and a C57BL/6 skin allograft, as described in Materials and
Methods. As expected (15), the percentage of
circulating DES+CD8+
alloreactive T cells fell dramatically early in the course of treatment
(Fig. 1
D). The percentage before treatment was 5.8 ±
0.9%; this fell to 0.6 ± 0.4% (range, 0.2–1.4%) on the day of
skin grafting. In contrast, levels of
DES+CD8+ T cells remained
essentially unchanged in untreated chimeras given skin allografts (Fig. 1B) and in chimeras given skin grafts and
anti-CD154 mAb monotherapy (Fig. 1C).
In mice treated with DST and anti-CD154 mAb, the percentage of
transgenic alloreactive
DES+CD8+ T cells appeared
to recover slowly over time (Fig. 1D). Compared with the
untreated control mice (Fig. 1A), levels of peripheral blood
DES+CD8+ T cells remained
statistically significantly lower in tolerized mice immediately after
the start of treatment and through 98 days (p
< 0.001 at each time point). After day 98, levels of
DES+CD8+ T cells remained
somewhat lower than, but statistically similar to, levels in untreated,
grafted controls.
Skin allografts in KB5 hemopoietic CBA chimeras tolerized with DST and anti-CD154 mAb survive uniformly until shortly after levels of DES+CD8+ T cells begin to recover
The median survival time (MST) of skin allografts on KB5
hemopoietic chimeras that were otherwise untreated (13 days,
n = 7) or treated only with a brief course of
anti-CD154 mAb monotherapy (15 days, n = 7) was
uniformly brief (Fig. 2A). In
contrast, MST of skin allografts in KB5 hemopoietic chimeras that were
treated with DST plus anti-CD154 mAb was substantially prolonged
(169 days, n = 12, p < 0.001). Nearly
half of the grafts survived for 200 days.
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B). Survival of skin allografts on wild-type CBA mice that
were treated with DST plus anti-CD154 mAb was substantially
prolonged (125 days, n = 31, p <
0.001) in comparison to CBA controls, and was statistically similar to
that of the KB5 chimeric recipients treated in the same way (169 days,
p = NS; Fig. 2A).
Inspection of Fig. 2 reveals that the rejection of skin allografts in
mice treated with DST and anti-CD154 mAb tended to occur largely
between 100 and 150 days after transplantation. Comparison with Fig. 1D reveals that this time frame coincides with the time
period during which levels of alloreactive
DES+CD8+ T cells in
treated mice began to approach, and were statistically
indistinguishable from, pretransplant levels.
The reappearance of DES+CD8+ T cells in tolerized mice could result from the expansion of DES+CD8+ T cells that had escaped deletion or from colonization of the periphery by new thymic emigrants. As a first step toward analyzing these possibilities, thymocytes were obtained from a separate cohort of otherwise untreated KB5 TCR-transgenic mice 79 days after bone marrow injection and analyzed for the presence of DES+ cells with an immature, i.e., CD4+CD8+, phenotype. We observed that of total CD4+CD8+ thymocytes, 2.3 ± 1.9% were also DES+ (n = 5).
Depletion of CD4+ T cells is associated with activation of alloreactive DES+CD8+ T cells and rejection of healed-in skin grafts in tolerized mice
Depletion of CD4+ T cells is known to cause
rapid rejection of successful healed-in skin allografts in
thymectomized mice treated with DST and anti-CD154 mAb
(18). We tested the hypothesis that depletion of
CD4+ T cells in graft-bearing animals leads to
activation of alloreactive CD8+ T cells. KB5
hemopoietic CBA chimeras were treated with DST and anti-CD154 mAb
and given a C57BL/6 skin allograft. A cohort of seven mice bearing
healed-in allografts for 50–59 days then received three consecutive
daily doses of anti-CD4 mAb (0.5 mg/dose). Consistent with our
previous report (18), all treated mice rejected their skin
grafts within 40 days (Fig. 2A). In contrast, only 1 of 12
(8%) of control mice not given anti-CD4 mAb rejected its graft
during that same interval of observation (Fig. 2A,
p < 0.001). To determine whether graft rejection was
accompanied by activation of alloreactive T cells, we measured the
percentage of
CD62LlowDES+CD8+
T cells obtained from a sample of the mice shown in Fig. 2A.
As shown in Table I, the percentage of
CD62LlowDES+CD8+
T cells was highest in the three mice undergoing graft rejection. The
percentage of activated
DES+CD8+ T cells tended to
be lower not only in mice with intact grafts, but also in mice that had
rejected grafts weeks earlier.
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We next assessed the role of anti-CD154 mAb concentration in the mechanism of allograft survival in mice treated with our standard protocol. To do so, we performed several analyses of Ab concentration and correlated them with two functional assays of costimulatory blockade and with skin graft survival. We tested the hypothesis that graft rejection occurs when residual concentrations of anti-CD154 mAb are too low to prevent costimulation and the generation of T-dependent Ab responses.
Normal C57BL/6 mice were selected at random and either left untreated
or given a single i.p. injection of the MR1 hamster anti-mouse
CD154 mAb at concentrations ranging from 0.016 to 0.5 mg. All mice also
received a single injection of SRBC, as described in Materials
and Methods. The concentration of hamster Ig was measured before
and 2, 7, and 14 days after injection. As shown in Fig. 3A, the absorption of Ab from
the peritoneal space occurred slowly, and was maximal by day
7.
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B, the titer in control mice that did not receive
anti-CD154 mAb became positive and maximal by day 7; the titer was
1:1280 in each of the three tested mice. Increasing doses of
anti-CD154 mAb were associated with decreasing hemagglutination
titers. The three mice treated with a single injection of 0.5 mg
anti-CD154 mAb generated only weakly positive titers. Comparison of
the data in Fig. 3, A and B, suggests that
substantial inhibition of anti-SRBC Ab production required the
presence of 100 µg/ml or more anti-CD154 mAb in the
circulation.
As an additional functional index of the inhibition of costimulatory
blockade, we also measured the concentration of mouse anti-hamster
Ig in these same mice. As shown in Fig. 3C, MR1
anti-CD154 mAb completely prevented the development of Abs to
itself when given at a dose of 0.5 mg. Comparing with the data in Fig. 3A, it appears that little or no anti-hamster IgG Ab
response was detectable when the concentration of anti-CD154 mAb
present was greater than 50 µg/ml.
Taken together, the data in Fig. 3, A—C, suggest that
concentrations of MR1 anti-CD154 mAb that are in the range of
50–300 µg/ml in vivo are required to block costimulation and
prevent T-dependent Ab responses.
We next determined the concentration of anti-CD154 mAb in
synchimeric mice that were actually treated with DST, four 0.5-mg
injections of anti-CD154 mAb, and a skin allograft. Ab
concentrations were determined serially beginning on day 14 after
grafting, which was day 10 after the final injection of Ab. As shown in
Fig. 3D, the concentration of Ab at this first time point
was 200 µg/ml and only declined to 50 µg/ml 50 days after
grafting. Comparison with Fig. 2A suggests that
concentrations of Ab insufficient to block costimulation occur at about
the time skin allografts begin to reject.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In those experiments, we analyzed mechanisms underlying not only the
induction, but also the maintenance or failure of peripheral tolerance
in euthymic mice treated with DST and anti-CD154 mAb. We have
previously documented that treatment with this protocol reproducibly
prolongs skin allograft survival in euthymic mice (5).
Tolerization is dependent on the presence of CD4+
cells and the deletion of CD8+ cells (15, 18). Although relatively nonantigenic islets generally survive
indefinitely, only 20% of skin allografts survive >100 days
(19). Permanent skin allograft survival in a majority of
recipients is achieved only if the graft recipients are thymectomized
(18), and even in thymectomized mice depletion of
CD4+ cells can induce graft rejection. Given
these data, we hypothesized that long-term maintenance of allografts in
tolerized recipients is a dynamic process that is dependent on the
number and activation state of alloreactive and regulatory cells.
To analyze this dynamic process quantitatively, we determined levels of therapeutic anti-CD154 mAb concentrations and correlated these levels with graft survival and circulating alloreactive CD8+ T cells in a new analytical system based on allo-TCR-transgenic hemopoietic chimeric graft recipients. We developed this model because it permitted the study of a trace population of high-affinity alloreactive T cells that not only circulated (28, 30), but also developed in a normal microenvironment. One might argue that these analyses could have been conducted using TCR-transgenic mice as recipients, but it is important to recall that TCR-transgenic mice do not generate normal immune responses due to severe skewing of their T cell repertoire (28, 30). One might also argue that the standard Jenkins single-transfusion chimera could have been used (30). This system has successfully been used to study the induction of transplantation tolerance (15), but again it should be recalled that standard Jenkins chimeras cannot be used to study the maintenance of tolerance in the context of newly released thymic emigrants. We designed our TCR-transgenic hemopoietic chimera to respond to tolerance induction in a manner similar to that observed in normal mice and to overcome the limitations inherent in both alternative model systems.
To validate our model system, we first documented that control KB5 TCR-transgenic hemopoietic chimeras reject allografts with normal kinetics. More importantly, we then documented that the median survival of skin allografts in KB5 chimeras tolerized with DST and anti-CD154 mAb was similar to that observed in tolerized normal CBA mice. As expected (15), the alloreactive DES+CD8+ T cells were deleted in the chimeras during tolerance induction. We also documented that TCR-transgenic T cells developed in the thymus and, over time, were able to repopulate the alloreactive DES+CD8+ T cell compartment in the peripheral tissues of the chimeras.
This TCR-transgenic hemopoietic chimera model system permitted us to examine the relationships among 1) newly released alloreactive CD8+ T cells in peripheral tissues, 2) levels of anti-CD154 mAb, and 3) regulation of alloreactive CD8+ T cell activation in the presence or absence of CD4+ cells in tolerized mice bearing healed-in skin allografts. Our findings in this system provide the first direct evidence that 1) alloreactive CD8+ T cells reappear in tolerant euthymic mice bearing intact allografts, and 2) graft rejection occurs in the context of increasing numbers of these alloreactive T cells in the absence of costimulation blockade.
Our data suggest specifically that the initiation of allograft rejection correlates with a decline in anti-CD154 mAb concentration to levels that are no longer sufficient to prevent costimulation. Concentrations less than 50 µg/ml were insufficient to prevent the generation of T-dependent Ab responses. This observation is of particular interest in light of our data demonstrating that graft rejection is also associated with the activation of CD8+ alloreactive cells. We (25) and others (26, 27) have shown that some of the T cells that develop in the thymus of mice bearing a healed-in allograft have not been tolerized to that graft. It has also been reported in nonhuman primates tolerized with anti-CD154 mAb that islet allograft maintenance requires periodic retreatment with the Ab (41). We interpret this set of observations to suggest that maintenance of allografts in tolerized recipients with healed-in grafts depends in part on levels of an anticostimulation reagent (in this case, anti-CD154 mAb) to prevent the activation of newly developed alloreactive T cells.
We suggest that the reappearance of alloreactive DES+CD8+ T cells in the chimeras is most likely the result of continuous reseeding of the periphery with new alloreactive CD8+ T cells. This interpretation is supported by our observation that CD4+CD8+DES+ immature T cells develop in the thymus of KB5 hemopoietic chimeras. We recognize that an alternative explanation for the reappearance of alloreactive CD8+ T cells could be the failure of our tolerance induction protocol to delete these cells completely; the residual cells could then simply reexpand in response to the allograft. We believe this to be unlikely, however, because the phenotype of most DES+ cells in tolerized chimeras was that of naive cells rather than activated or memory cells. Only after CD4+ T cells had been deliberately deleted and skin allografts were undergoing rejection did we detect activated DES+CD8+ T cells.
Our data demonstrate that graft rejection was accompanied by activation of alloreactive T cells, but we were unable to correlate percentages of DES+CD8+CD62Llow peripheral blood cells with impending graft rejection (data not shown). We speculate that the lack of predictive power associated with this phenotype is due to rapid emigration from the blood of activated cells to nonlymphoid tissues (42), presumably including the graft itself.
Another alternate explanation for the reappearance of alloreactive CD8+ T cells could be the redistribution of DES+ cells to tissues that we did not analyze; it could be argued that over time such cells then reverted to a naive phenotype (43). If this were the case, however, the DES+CD8+ T cells that had migrated to the lymphoid compartment from sites of sequestration would again be expected to exhibit an activated phenotype. The present data do not exclude this possibility, but in a preliminary experiment we attempted to detect DES+CD8+ cells in KB5 hemopoietic chimeras that had been thymectomized just before tolerance induction and deletion of preexisting alloreactive CD8+ T cells; no DES+CD8+ cells were detectable for up to 97 days (T. G. Markees, unpublished observations).
Although the majority of high-affinity alloreactive DES+CD8+ T cells seem to disappear from the circulation within 3 days (15), we are still left to account for the rejection of healed-in grafts in thymectomized recipients treated with anti-CD4 mAb (18). To account for this observation, we would point out that our data document the deletion of a clonal, high-affinity trace cell population, specifically the DES+CD8+ cells. Allograft rejection, however, represents a polyclonal immune response that also involves lower affinity cells, some of which are likely to escape deletion. Our documentation that the DES+CD8+ cells are deleted allows us to hypothesize that residual low-affinity alloreactive cells are the agents of graft rejection in tolerized thymectomized mice treated with anti-CD4 mAb. In this regard, it will be of interest to determine in future studies whether the number of CD4+ regulatory cells in tolerized recipients is stable or declines over time.
In conclusion, we have developed a new model system for the study of transplantation tolerance. The key element in this system is the presence of trace populations of high-affinity alloreactive CD8+ T cells in recipient mice that are amenable to tolerance induction based on costimulation blockade. The trace populations of CD8+ alloreactive T cells in this system are self-renewing and mature in a normal immunological environment, making it possible for the first time to analyze the mechanisms that determine whether healed-in allografts are maintained or rejected. Using these KB5 TCR-transgenic synchimeric mice tolerized with DST and anti-CD154 mAb, we have documented that the maintenance of healed-in allografts is a dynamic process dependent on 1) anti-CD154 mAb concentration, 2) CD4+ regulatory cells, and 3) activated alloreactive CD8+ thymic emigrants that have repopulated the periphery after tolerization. We hypothesize that these three factors comprise the core determinants of allograft maintenance in tolerized recipients. The model system we have described should now permit the dissection of each of these factors and their interactions.
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Acknowledgments |
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Footnotes |
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2 The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.
3 Current address: Harvard School of Public Health, Boston, MA 02115.
4 Address correspondence and reprint requests to Dr. Aldo A. Rossini, Diabetes Division, University of Massachusetts Medical School, 373 Plantation Street, Biotech 2, Suite 218, Worcester, MA 01605. E-mail address: aldo.rossini{at}umassmed.edu
5 Abbreviations used in this paper: DST, donor-specific transfusion; MST, median survival time; OPD, o-phenylene diamine.
Received for publication August 2, 2001. Accepted for publication September 19, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and CTLA4. J. Clin. Invest. 101:2446.[Medline]
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 M. A. Brehm, J. Mangada, T. G. Markees, T. Pearson, K. A. Daniels, T. B. Thornley, R. M. Welsh, A. A. Rossini, and D. L. Greiner Rapid quantification of naive alloreactive T cells by TNF-{alpha} production and correlation with allograft rejection in mice Blood, January 15, 2007; 109(2): 819 - 826. [Abstract] [Full Text] [PDF]
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T. B. Thornley, M. A. Brehm, T. G. Markees, L. D. Shultz, J. P. Mordes, R. M. Welsh, A. A. Rossini, and D. L. Greiner TLR Agonists Abrogate Costimulation Blockade-Induced Prolongation of Skin Allografts J. Immunol., February 1, 2006; 176(3): 1561 - 1570. [Abstract] [Full Text] [PDF]
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 M. Sho, K. Kishimoto, H. Harada, M. Livak, A. Sanchez-Fueyo, A. Yamada, X. X. Zheng, T. B. Strom, G. P. Basadonna, M. H. Sayegh, et al. Requirements for induction and maintenance of peripheral tolerance in stringent allograft models PNAS, September 13, 2005; 102(37): 13230 - 13235. [Abstract] [Full Text] [PDF]
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 E. J. Gordon, L. S. Wicker, L. B. Peterson, D. V. Serreze, T. G. Markees, L. D. Shultz, A. A. Rossini, D. L. Greiner, and J. P. Mordes Autoimmune Diabetes and Resistance to Xenograft Transplantation Tolerance in NOD Mice Diabetes, January 1, 2005; 54(1): 107 - 115. [Abstract] [Full Text] [PDF]
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T. Pearson, P. Weiser, T. G. Markees, D. V. Serreze, L. S. Wicker, L. B. Peterson, A.-M. Cumisky, L. D. Shultz, J. P. Mordes, A. A. Rossini, et al. Islet Allograft Survival Induced by Costimulation Blockade in NOD Mice Is Controlled by Allelic Variants of Idd3 Diabetes, August 1, 2004; 53(8): 1972 - 1978. [Abstract] [Full Text] [PDF]
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A. van Maurik, B. F. de St. Groth, K. J. Wood, and N. D. Jones Dependency of Direct Pathway CD4+ T Cells on CD40-CD154 Costimulation Is Determined by Nature and Microenvironment of Primary Contact with Alloantigen J. Immunol., February 15, 2004; 172(4): 2163 - 2170. [Abstract] [Full Text] [PDF]
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A. A. Rossini Autoimmune Diabetes and the Circle of Tolerance Diabetes, February 1, 2004; 53(2): 267 - 275. [Abstract] [Full Text]
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T. Pearson, T. G. Markees, D. V. Serreze, M. A. Pierce, M. P. Marron, L. S. Wicker, L. B. Peterson, L. D. Shultz, J. P. Mordes, A. A. Rossini, et al. Genetic Disassociation of Autoimmunity and Resistance to Costimulation Blockade-Induced Transplantation Tolerance in Nonobese Diabetic Mice J. Immunol., July 1, 2003; 171(1): 185 - 195. [Abstract] [Full Text] [PDF]
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N. E. Phillips, T. G. Markees, J. P. Mordes, D. L. Greiner, and A. A. Rossini Blockade of CD40-Mediated Signaling Is Sufficient for Inducing Islet But Not Skin Transplantation Tolerance J. Immunol., |
