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* Department of Surgery and Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Medical Center, Pittsburgh, PA 15213; and
Department of Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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11
were deleted selectively. Immunocompetence of tolerant FL-SC-treated
mice was proven by rapid rejection of third-party skin grafts. To our
knowledge this is the first report that mobilization of DC in donor
cell infusions can be used to induce skin graft tolerance across MHC
barriers, accompanied by specific deletion of donor-reactive T
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In transplantation, it is well recognized that injection of unmodified donor hemopoietic cells (including DC) into immunocompromised adult hosts, with consequence establishment of mixed chimerism, can lead to alloantigen-specific tolerance (10, 11, 12). Thus, for example, i.v. infusion of allogeneic spleen cells (SC), with or without donor bone marrow cells (BMC), followed by cyclophosphamide (CP), can induce long-lasting skin graft survival in minor histocompatibility complex-disparate, but not MHC-disparate, mouse strain combinations (13, 14). The efficacy of this regimen has been ascribed to the elimination of proliferating alloantigen-specific T cells induced by donor APC (15).
We hypothesized that more potent/extensive activation of alloreactive T cell clones by donor cell infusions containing enhanced numbers of in vivo stimulatory DC, followed by their destruction, might enhance the tolerogenicity of the donor cell/CP regimen. The endogenous ligand for Flt3 (FMS-like tyrosine kinase 3 ligand (FL)) that has been employed recently to study the effect of DC on transplant outcome (16, 17) was used to dramatically enhance stimulatory DC in donor hemopoietic tissue (18, 19). We then ascertained the influence of priming with FL-mobilized SC on CP-mediated deletion of donor-reactive T cells and skin transplant survival across MHC barriers. Our data show that indefinite, donor-specific skin graft tolerance (100 days) can be induced in recipients of FL-mobilized donor cells, but not in mice given normal SC. Tolerance was associated with persistent blood macrochimerism and with selective deletion of peripheral donor-reactive T cells. Thus, mobilization of immunostimulatory donor DC in hemopoietic cell infusions can be used to promote robust donor-specific transplant tolerance across MHC barriers.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Age-matched female C57BL/10SnJ (B10; H2Kb, IAb, IE-), B10.Br/Sg SnJ (B10.BR; H2Kk, IAk, IE+), and B10.D2 (H2Dd, IAd, IE+) mice, 8–12 wk of age, were purchased from The Jackson Laboratory (Bar Harbor, ME). They were maintained in the specific pathogen-free facility of the University of Pittsburgh Medical Center in accordance with institutional guidelines.
Reagents
Recombinant mouse GM-CSF was provided by Dr. S. K. Narula
(Schering-Plough Research Institute, Kenilworth, NJ). FITC-, PE-, or
CyChrome-conjugated mAbs used to detect cell surface expression of CD4
(H129.19), CD11c (HL3), CD40 (HM40–3), CD45 (30-F11), CD54 (ICAM-1;
3E2), CD80 (16-10A1), CD86, (GL1), H2Kb
(AF6-88.5), H2Dd (34-2-12),
IAd (AMS-32.1), V 8.1/8.2 TCR (MR5-2), and
V11 TCR (RR3-15) by flow cytometry as well as isotype-matched
control irrelevant mAbs were purchased from BD PharMingen (San Diego,
CA). Cyclophosphamide (CP; Neosar) was obtained from Pharmacia & Upjohn
(Kalamazoo, MI).
In vivo mobilization of DC-enriched donor SC by FL
B10.D2 donor mice received either no treatment or Chinese hamster ovary cell-derived recombinant human FL (Immunex Corporation, Seattle, WA) in sterile PBS (10 µg/mouse/day, i.p.) for 10 consecutive days.
Preparation, culture and analysis of SC preparations
Following RBC lysis, SC were either resuspended in sterile PBS for i.v. injection or cultured in RPMI 1640 (Life Technologies, Grand Island, NY) containing 10% (v/v) heat-inactivated FBS, 2 mM L-glutamine, 50 U/ml penicillin and streptomycin, 2 mM nonessential amino acids (all reagents from Life Technologies; subsequently referred to as complete medium), and 50 U/ml recombinant mouse GM-CSF to maintain viability for 16 h at 37°C in 5% CO2 in air. SC suspensions from normal or FL-mobilized mice were analyzed either fresh or after 16-h culture for surface expression of CD11c, CD40, CD54, CD80, CD86, and MHC class II by flow cytometry, as previously described (20).
Conditioning of skin graft recipients
B10 mice were given 108 normal SC or DC-enriched SC from FL-treated, H2-mismatched, B10.D2 donor mice i.v. on day 0, 200 mg/kg i.p. CP on day 2, and 107 T cell-depleted BMC from normal B10.D2 donors on day 3.
Skin transplantation
Skin grafting was performed on day 14, as previously described (21). Briefly, square full-thickness skin grafts (1 cm2) were prepared from the trunk skin of donors. Graft beds (1 cm2) were prepared on the right lateral thoracic wall of the recipient mice. The graft was fixed to the graft bed with eight interrupted sutures of 5-0 silk thread and covered with protective tape. The first inspection was conducted 7 days postgrafting, followed by daily inspection. Grafts were considered as rejected at the time of complete sloughing or when they formed a dry scar. Survival was expressed as the mean survival time ± 1 SD.
Mixed leukocyte reaction
Splenic T cells were purified by passage through nylon wool columns, then used as responders (2 x 105/well in round-bottom, 96-well plates) against graded numbers of gamma-irradiated (20 Gy), freshly isolated, B10.D2 (donor) or B10.BR (third-party) splenocytes in one-way MLR. Cultures were maintained in complete medium for 72 h at 37°C in 5% CO2 in air. For the final 18 h individual wells were pulse-labeled with 1 µCi [3H]thymidine. Results are expressed as the mean cpm ± 1 SD.
CTL assay
Splenocytes from recipients of B10.D2 SC were restimulated in vitro for 5 days with gamma-irradiated (20 Gy) B10.D2 donor or B10.BR third-party SC at a 1/1 ratio before use as effectors in CTL assays. The P815 (H2d) mastocytoma (TIB64; American Type Culture Collection, Manassas, VA) and the R1.1 (H2k) lymphoma cell lines (TIB42; American Type Culture Collection) were used as specific and third-party targets, respectively. Target cells were labeled with 100 µCi Na251CrO4 (NEN, Boston, MA) and plated at 5 x 103/well in 96-well plates. Serial 2-fold dilutions of effector cells were added to give E:T cell ratios of 100:1, 50:1, 25:1, and 12.5:1. Following 4-h incubation at 37°C in 5% CO2 in air, specific 51Cr release was determined. The percent cytotoxicity was calculated using the formula: % specific cell lysis = 100 x [experimental (cpm) - spontaneous (cpm)/maximum (cpm) - spontaneous (cpm)]. Results are expressed as the mean ± 1 SD percentage of 51Cr released in triplicate cultures.
Analysis of chimerism and TCR V families
Donor cell chimerism and TCR V families were analyzed in
peripheral blood samples by flow cytometry. Briefly, RBC were lysed
with 0.83% (w/v) NH4Cl and lymphocytes were
enriched by density gradient centrifugation (Lymphocyte-M; Cedarlane
Laboratories, Hornby, Canada) according to the manufacturer’s
instructions. Thereafter, 1–2 x 105 cells
were blocked with 10% (v/v) normal goat serum for 10 min at 4°C,
then stained with mAbs against donor or recipient MHC class I
(H2Dd and H2Kb,
respectively) or against V8.1/8.2 or V11 TCR and CD4 for 30 min
at 4°C. Cells were costained with mAb against CD45 (common leukocyte
Ag) to exclude nonspecific staining of residual erythrocytes.
Appropriate isotype-matched Igs were used as negative controls. After
staining, the cells were fixed with 2% (w/v) paraformaldehyde, then
analyzed using an EPICS Elite flow cytometer (Beckman Coulter,
Hialeah, FL).
Statistics
Results are expressed as the mean ± 1 SD. Values were compared using Student’s t test for independent samples. Normal distribution of values, a prerequisite for using the t test, was proved using the Kolmogorov-Smirnov test. A value of p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first examined the impact of systemic FL administration on the
incidence and phenotype of CD11c+ DC in freshly
isolated B10.D2 SC suspensions. Single and multicolor immunostaining,
followed by flow cytometric analysis, were performed to ascertain the
incidence of CD11c+ DC expressing various
differentiation markers. After 10 days of FL administration, an 
20-
to 25-fold increase in CD11c+ DC was detected
consistently in FL-SC compared with normal control SC (Fig. 1
a). In freshly isolated
FL-SC, the expression of CD40, CD80, CD86, and MHC class II on
CD11c+ cells was low to moderate (Fig. 1b), indicating that the DC present were at an immature
stage of differentiation. However, after overnight (16-h) culture of
FL-SC in the absence of exogenous cytokines, the expression of these
Ags was increased markedly (Fig. 1b), indicating that the DC
in freshly isolated FL-SC matured readily in vitro.
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To test the stimulatory activity of B10.D2 FL-SC in vivo,
recipient B10 mice were primed i.v. with 107
freshly isolated SC from either normal control or FL-treated B10.D2
mice. Seven days later, recipient splenocytes were restimulated with
donor or third-party (B10.BR) SC. As shown in Fig. 1
c, T
cells from B10 mice primed with B10.D2 FL-SC responded more vigorously
to donor than those primed with normal B10.D2 SC. By contrast, the
weaker responses to third-party (B10.BR) alloantigens were almost
identical in the two groups (Fig. 1c). These results clearly
indicated that in vivo FL-SC containing enhanced numbers of donor DC
were more potent allogeneic T cell stimulators than normal SC and
primed recipient T cells in a donor-specific manner.
FL-SC modestly prolong skin allograft survival when combined with CP
We then examined the influence of donor FL-SC on allograft
survival. B10 mice were primed with either 108
FL-SC or normal SC from B10.D2 donors, followed 2 days later by a
single injection of CP. Skin grafting from normal donor mice was
performed 14 days thereafter. As shown in Fig. 2a, FL-SC prolonged graft
survival more effectively than normal SC when combined with CP
treatment. However, indefinite skin graft survival was achieved in only
10% of mice given FL-SC in this fully MHC-mismatched strain
combination and in no mice given normal SC.
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We have shown recently that the addition of T cell-depleted BMC 1
day after CP and busulfan administration prolongs skin allograft
survival (22). Thus, in an effort to further enhance graft
survival, donor BMC were injected 1 day after CP. B10 mice given
108 FL-SC from B10.D2 mice on day 0, CP on day 2,
and then 107 T cell-depleted BMC from normal
B10.D2 mice on day 3, exhibited long-lasting (100 day) skin graft
survival (Fig. 2b). By contrast, B10.D2 grafts in all B10
mice given normal B10.D2 SC, CP, and BMC, were rejected within 45 days
(Fig. 2b). Graft prolongation was donor specific, as
third-party B10.BR (H2k) skin was rejected
acutely (Fig. 2c). To ascertain whether robust
transplantation tolerance had been induced, second-set donor skin
grafts were performed in B10 recipients of B10.D2 FL-SC, CP, and normal
B10.D2 BMC that had accepted B10.D2 skin 100 days. As shown in Fig. 2d, these mice accepted donor-specific B10.D2 skin grafts
for 100 days, but rapidly rejected third party (B10.BR) grafts. Thus,
the regimen of donor FL-SC, CP, and BMC induced robust, donor-specific,
skin graft tolerance across MHC barriers.
Skin graft tolerance is accompanied by stable mixed chimerism
Chimerism was assessed in B10 graft recipients by flow cytometric
analysis of donor (H2Dd+) cells in the peripheral
blood 5 and 10 wk after skin grafting. Stable mixed chimerism was
established consistently only in mice treated with B10.D2 FL-SC, CP,
and B10.D2 BMC (Fig. 3a and
Table I, group 12), and accompanied
long-lasting skin graft tolerance (Fig. 2b). In B10 mice
treated with normal B10.D2 SC, CP, and B10.D2 BMC, mixed chimerism was
observed in only a minor proportion of the animals, and the degree of
chimerism observed was much lower in comparison (Fig. 3a and
Table I, group 11). In B10 mice given normal B10.D2 SC and CP, no
chimerism was observed (Table I, group 5).
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To further investigate the underlying nature of the tolerance
induced in B10 mice by B10.D2 FL-SC, CP, and B10.D2 BMC, anti-donor
MLR and CTL activity were examined 6 wk after tolerance induction. Host
splenocytes were examined for their capacity to generate CTL activity
against B10.D2 or B10.BR (third-party) Ags in one-way MLR (Fig. 4a). Anti-donor CTL activity
was profoundly suppressed in mice treated with B10.D2 FL-SC, CP, and
B10.D2 BMC, but not in recipients of normal B10.D2 SC, CP, and B10.D2
BMC. All groups generated CTL responses to third-party Ags (Fig. 4a). Similar results were observed in the MLR assay (Fig. 4b). Thus, split tolerance (ex vivo anti-donor T cell
reactivity in the presence of functional in vivo tolerance) was not
evident using the induction protocol we adopted.
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To investigate the peripheral T cell repertoire in the tolerant
animals, we examined TCR V11 and V8 expression on blood T cells
in the chimeric B10 (IE-) recipients of B10.D2
(IE+) skin (Fig. 3b and Table II). In untreated B10 mice,
CD4+ V11+ T cells were
readily detected (Fig. 3b, and Table I, group 1) whereas
these cells were very infrequent in B10.D2 mice (Fig. 3b and
Table II, group 13). In all skin-allografted chimeric mice given B10.D2
FL-SC, CP, and B10.D2 BMC, V11+ T cells were
markedly and significantly reduced at 5 and 10 wk (Fig. 3b
and Table II, group 12). This reduction in T cells was specific, since
V8+ T cells were not altered significantly
(Table II). V11+ T cells were not reduced in
any other experimental group (Table II, groups 2–11).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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20- to 25-fold increase
compared with normal spleen) can be used to promote the induction of
robust, donor-specific skin transplant tolerance. Thus, 100 day skin
graft survival was achieved consistently in fully MHC-mismatched
recipients also given a single dose of CP and T cell-depleted donor BMC
before grafting. Donor-specific tolerance was associated with durable
macrochimerism and the selective deletion of donor-reactive T cells.
These findings contrast markedly with the failure of normal donor SC to
promote skin graft tolerance across both MHC and minor
histocompatibility barriers using an otherwise identical treatment
regimen in this and previous studies (23, 24). Thus,
although they were prolonged significantly, no skin grafts survived
45 days in recipients of normal donor SC, CP, and BMC in the present
study. Similar regimens using normal donor SC plus CP and BMC have also
failed to achieve tolerance in fully allogeneic rat renal graft
recipients (23), although split tolerance to fully
allogeneic liver grafts (i.e., in vivo tolerance/graft acceptance, but
ex vivo anti-donor T cell reactivity) has been reported
(25). In the latter instance it was concluded that the
combination of normal SC and CP was insufficient to completely
destroy/anergize donor-reactive T cells.
The FL-mobilized SC that promoted skin graft tolerance were
characterized by markedly enhanced numbers of functional
CD11c+ DC. As we have shown, overnight (18-h)
incubation of these cells resulted in marked up-regulation both of
surface MHC class II and costimulatory molecules, indicative of
spontaneous transition to a mature, T cell stimulatory phenotype. FL
administration induces elevated numbers of both
CD11c+CD8
-CD11b+
(myeloid) and
CD11c+CD8+CD11b-
(lymphoid-related) splenic DC subsets (8). After overnight
incubation, each subset elicits potent naive T cell proliferative
responses in vitro and primes allogeneic T cells in vivo with equal
high efficiency (8). Consistent with the marked
enhancement of this functional DC component, freshly isolated FL-SC
were much more effective than normal SC in priming naive allogeneic
hosts on a per cell basis. This was evidenced by striking increases in
ex vivo host T cell proliferative responses to donor, but not to
third-party stimulators 7 days after FL-SC infusion (Fig. 1, c–e). Future studies that compare the influence of purified
DC (including DC subsets) from FL-treated and normal mice on skin
allograft survival are likely to provide mechanistic insight.
Conceivably, because of their enhanced T cell stimulatory activity,
overnight cultured/matured DC may be more effective than freshly
isolated DC.
We speculated that destruction/deletion of markedly expanded clones of alloreactive T cells in FL-SC recipients by the alkylating agent CP might prove more effective in promoting tolerance than had been achieved previously in recipients of normal SC. In addition, use of a single dose of CP constituted a non-irradiation-based conditioning approach for deletion of (early) hemopoietic stem cells and subsequent engraftment of donor stem cells. It also avoided the peripheral toxicities associated with use of gamma irradiation and/or depletion of the peripheral immune system (10, 11, 26, 27). Others have shown recently that another alkylating agent, busulfan, in combination with costimulation blockade can promote high level chimerism and fully allogeneic skin graft acceptance using (as in the present study) clinically relevant numbers of T cell-depleted donor BMC (28). The engrafting doses of T cell-depleted donor BMC used in both the latter study and the present work are substantially lower than those (clinically impractical) multiple or mega doses of BMC employed in other recent reports without recipient cytoreductive conditioning (29, 30).
The capacity of in vivo-mobilized immunostimulatory donor DC together
with CP to promote lasting hemopoietic chimerism and robust skin
allograft tolerance appears to reflect specific deletion of
alloactivated (superantigen-specific
CD4+V11+) T cells
normally deleted in the donor strain. Although previously the infusion
of donor DC before transplantation has been shown to promote long term
allograft survival in unmodified recipients, this effect has been
attributed to the immaturity of the donor DC at the time of their
infusion and to their ability to induce T cell hyporesponsiveness.
Costimulation blockade that interferes with their potential T cell
allostimulatory activity in vivo further enhances the tolerogenic
potential of these immature DC and increases apoptotic death of
alloreactive T cells in graft recipients (31, 32). By
contrast, as we have shown here that infusion of FL-mobilized SC
comprising 20% donor DC that potently prime alloreactive T cells in
vivo, facilitates CP-mediated destruction/deletion of these activated T
cell clones. In the absence of CP (FL-SC plus BMC administration
alone), neither deletion of alloactivated T cells, persistent chimerism
(at 5 wk), nor prolongation of graft survival was observed. This
indicated that the potent T cell immunostimulatory ability of
FL-mobilized donor DC alone was insufficient to achieve these effects.
Indeed, it is well recognized that in normal hosts, infusion of mature
allostimulatory donor DC before transplantation accelerates graft
rejection (6).
In conclusion, we have shown that donor DC mobilization combined with CP and T cell-depleted donor BMC, promotes persistent (high level) hemopoietic chimerism and robust transplant tolerance in a fully MHC-incompatible mouse strain combination. As with other successful chimerism induction protocols, we have observed donor-specific tolerance to secondary skin grafts, specific deletion of superantigen-specific T cells, and the absence of ex vivo anti-donor T cell proliferative and cytotoxic activity. These findings reveal a novel, tolerance-promoting property of growth factor-mobilized, in vivo immunostimulatory DC. Further investigation of this model, and in particular whether reduced levels/alternative forms of myelosuppression can retain both the chimerism- and tolerance-inducing effect, will establish the potential utility of this approach in both bone marrow (hematologic disease) and organ transplantation.
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Acknowledgments |
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Footnotes |
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2 M.E. and H.H. contributed equally to this paper.
3 Current address: Department of Urology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
4 Address correspondence and reprint requests to Dr. Angus W. Thomson, University of Pittsburgh Medical Center, 200 Lothrop Street, W1544 BSTWR, Pittsburgh, PA 15213. E-mail address: thomsonaw{at}msx.upmc.edu
5 Abbreviations used in this paper: DC, dendritic cell; BMC, bone marrow cell; CP, cyclophosphamide; FL, Flt3 ligand; SC, spleen cell.
Received for publication April 26, 2002. Accepted for publication June 25, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+ dendritic cells prolong the survival of vascularized heart allografts. J. Immunol. 168:143.6+ T cells in cyclophosphamide-induced tolerance to H-2-compatible, Mls-disparate antigens. J. Exp. Med. 171:97.This article has been cited by other articles:
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  U. I. Chaudhry, S. C. Katz, T. P. Kingham, V. G. Pillarisetty, J. R. Raab, A. B. Shah, and R. P. DeMatteo In vivo overexpression of Flt3 ligand expands and activates murine spleen natural killer dendritic cells FASEB J, May 1, 2006; 20(7): 982 - 984. [Abstract] [Full Text] [PDF]
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 M. Harano, M. Eto, T. Iwai, K. Tatsugami, K. Kiyoshima, Y. Kamiryo, M. Tsuneyoshi, Y. Yoshikai, and S. Naito Renal Cancer Treatment with Low Levels of Mixed Chimerism Induced by Nonmyeloablative Regimen Using Cyclophosphamide in Mice Cancer Res., November 1, 2005; 65(21): 10032 - 10040. [Abstract] [Full Text] [PDF]
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 Z. Wang, A. Castellaneta, A. De Creus, W. J. Shufesky, A. E. Morelli, and A. W. Thomson Heart, but Not Skin, Allografts from Donors Lacking Flt3 Ligand Exhibit Markedly Prolonged Survival Time J. Immunol., May 15, 2004; 172(10): 5924 - 5930. [Abstract] [Full Text] [PDF]
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