Allosuppressive Donor CD+CD+ Regulatory T Cells Detach from the Graft论文 and Circulate in Recipients after Liver Transplantation
【摘要】 organ transplantation (tx) results in a transfer of donor leukocytes from the graft to the recipient, which can lead to chimerism and may promote tolerance. it remains unclear whether this tolerance involves donor-derived regulatory t cells (tregs). in this study, we examined the presence and allosuppressive activity of cd4+cd25+foxp3+ tregs in perfusates of human liver grafts and monitored the cells presence in the circulation of recipients after liver tx. vascular perfusions of 22 liver grafts were performed with university of wisconsin preservation and albumin solutions. flow cytometric analysis revealed that perfusate t cells had high lfa-1 integrin expression and had a reversed cd4 to cd8 ratio compared with control blood of healthy individuals. these findings indicate that perfusate cells are of liver origin and not derived from residual donor blood. further characterization of perfusate mononuclear cells showed an increased proportion of cd4+cd25+ctla4+ t cells compared with healthy control blood. increased percentages of foxp3+ cells, which were negative for cd127, confirmed the enrichment of tregs in perfusates. in mlr, cd4+cd25+ t cells from perfusates suppressed proliferation and ifn- production of donor and recipient t cells. in vivo within the first weeks after tx, up to 5% of cd4+cd25+ctla4+ t cells in recipient blood were derived from the donor liver. in conclusion, a substantial number of donor tregs detach from the liver graft during perfusion and continue to migrate into the recipient after tx. these donor tregs suppress the direct pathway alloresponses and may in vivo contribute to chimerism-associated tolerance early after liver tx.
【关键词】 allosuppressive regulatory circulate recipients transplantation
organ transplantation (tx)3 results in a transfer of leukocytes present in the graft into the recipient. the migration of donor cells to host tissues results in chimerism and this response has been proposed to be associated with donor-specific tolerance (1, 2). the relevance of these passenger leukocytes in tx outcome has been demonstrated in several models showing significant roles in regulation of immune reactions after organ tx. in a heart tx model, selective ab-mediated depletion of donor leukocytes leads to the prevention of tolerance induction and is associated with severe acute or chronic graft rejection (3). similar results have been found in experimental liver tx, in which depletion of passenger leukocytes leads to rejection of the graft in spontaneously tolerant recipients (4, 5). reconstitution of donor leukocytes by i.v. injection recovers graft acceptance. part of this recovery has been shown to be dependant on t cells, but not b cells or monocytes/macrophages, as shown by selective deletion from the reconstituting donor leukocytes (6, 7). however, the exact mechanism of this so-called chimerism-associated tolerance remains unclear. it has been suggested that high levels of chimerism, like for instance following bone marrow tx after myeloablation, are associated with tolerance through clonal deletion or anergy induction of host t cells. low levels of chimerism, such as following solid organ tx, are associated with tolerance through regulatory mechanisms that may involve active suppression of alloresponses by regulatory t cells (tregs) (8, 9). both in clinical and experimental tx, chimerism and chimerism-associated tolerance are more frequently seen with liver compared with other organ tx (10, 11). higher levels of chimerism in liver tx are associated with reduced incidences of acute rejection and better initial graft acceptance (12).
within the regulatory cell populations, cd4+cd25+ tregs play a critical role in various models of transplant tolerance (13, 14, 15, 16). regulation of alloresponses seems to depend in part on ctla-4, which is constitutively expressed by tregs (17, 18). bigenzahn et al. (19) showed that depletion of cd25+ cells shortly after nonmyeloablative bone marrow tx and costimulation blockade significantly reduced tolerance induction. this finding suggests that cd4+cd25+ tregs may be actively involved in chimerism-associated tolerance, particularly early after tx. however, this involvement remains controversial because other studies did not confirm a role for cd4+cd25+ tregs after bone marrow tx (20, 21).
to determine the specific role of donor leukocytes in the outcome of solid organ tx, it is important to phenotypically (22) and functionally characterize donor-derived leukocytes. in clinical liver tx, the effluent solution passing through the graft livers during perfusion before tx has been shown to be useful for this purpose. the leukocytes isolated from this perfusate solution represent detached liver-associated leukocytes, as shown by increased proportion of cd8+ cells that outnumber cd4+ cells and high nk cell numbers comparable with cell numbers in liver tissue (23, 24). furthermore, the myeloid dendritic cell (dc) population present in the perfusates has an immature phenotype identical with dc isolated from liver tissue and produced higher amounts of il-10 compared with blood dc (24).
in the current study, we determined the migration and suppressive capacity of donor cd4+cd25+ tregs transferred from the liver graft into the perfusate and the recipient after tx. we show that the lymphocyte population that migrates from the human liver is enriched for cd4+cd25+ forkhead box p3 (foxp3)+ cells and that these cells suppress proliferation and ifn- production of recipient t cells in vitro. in addition, we demonstrate that donor-derived cells with a regulatory phenotype can be detected in substantial numbers in the circulation of recipients after liver tx.
materials and methods
perfusate and pbmc collection
liver perfusates were collected from 22 human liver grafts. during the backtable procedure, the grafts were perfused through the portal vein with 1c2 l of university of wisconsin solution to remove residual blood from the vasculature. immediately before tx, the donor liver was perfused with 200 up to 500 ml of human albumin solution under hydrostatic pressure, and the perfusate was collected from the vena cava. mononuclear cells from perfusate were isolated within 12 h by density gradient centrifugation using ficoll paque plus (amersham biosciences). pbmc were obtained from healthy volunteers (n = 14), which served as control. after isolation, cells were stored in 10% dctla-4-pe, cd3-fitc, cd3-pe, and igg2a-pe from immunotech; foxp3-allophycocyanin, cd127-fitc, and isotype igg2a-allophycocyanin from ebioscience; cd11a from biosource international; and secondary ab (goat anti-mouse) fitc, cd8-allophycocyanin, and cd4-allophycocyanin from dakocytomation. hla-a2 staining was performed with an anti-hla-a2 ab derived from a hybridoma (clone bb7.2, no. hb-82; american type culture collection) followed by an fitc-conjugated rabbit anti-mouse igg secondary ab (dakocytomation).
flow cytometric analysis
after thawing, liver perfusate mononuclear cells (lpmc) and pbmc were washed twice with pbs containing 0.3% bsa followed by staining with primary mabs cd3, cd4, cd8, cd25, and cd127 in pbs/0.3% bsa (30 min at 4?c). following primary incubation, cells were washed, and for staining of intracellular ctla-4 or foxp3, the cells were fixed and permeabilized using the intraprep reagents (immunotech) or fixation/permeabilization agents supplied by ebioscience, respectively. hla-a2-positive cells were determined by incubation with anti-hla-a2 ab followed by staining with fitc-conjugated anti-mouse igg. expression of lfa-1 (cd11a) was determined in a similar manner by primary staining for cd11a followed by secondary staining with fitc-conjugated anti-mouse igg. flow cytometric analysis was performed using facscalibur and cellquest pro software (bd biosciences).
cd4+cd25+ t cell isolation
cd4+ cells were purified from fresh lpmc and pbmc using the untouched cd4+ t cell isolation kit (miltenyi biotec). after washing with pbs/0.3% bsa, cd4+ t cells were incubated with anti-cd25 microbeads (miltenyi biotec) followed by a positive selection of cd4+cd25+ t cells, according to the manufacturer?s instructions. the cd4+cd25c fraction was used as responder cells. the purified treg fraction contained >90% pure cd4+cd25+ t cells.
mlr and suppressor activity assays
responder cd4+cd25c t cells of recipient and donor were labeled with 2 m cfse (molecular probes), and 1.0 x 105 cells/well were stimulated with irradiated (5 gy) donor lpmc (5 x 104 cells) and recipient pbmc (5 x 104 cells) in a 96-well round-bottom plate. cells were cultured in rpmi 1640 with l-glutamine (cambrex bioscience) supplemented with 10% pooled heat-inactivated human serum, 100 iu/ml penicillin, and 100 g/ml streptomycin in a total volume of 200 l. to determine the suppressive activity of cd4+cd25+ t cells isolated from lpmc and pbmc, increasing numbers of cd4+cd25+ t cells (1.0 and 3.0 x 104) were added to the cultures. cultures were performed in duplicate or triplicate. at day 4 of culture, 100 l of culture medium was replaced, and the concentration of ifn- was measured by elisa (u-cytech). after 5 days, t cell divisions were analyzed by flow cytometry by staining the cells with cd3 and cd4 abs. cfse flow cytometry data were analyzed by modfit software version 3.0 (verity software house). the proliferation index, which is the sum of the cells in all generations divided by the computed number of original parent cells, indicates the extent of t cell expansion. if the proliferation index is equal to one, than no t cell division took place during the course of the culture.
for the mlr and ifn- production, statistical analysis was performed by analysis of the logarithmic transformation of the dependent variable with random intercept and random slope using proc mixed in sas version 9.1 (sas institute). significant differences between lpmc and pbmc flow cytometric results were determined with the mann-whitney u test using spss software version 11.0.
lpmc are of liver origin
perfusates were collected during the pre-tx albumin perfusion of donor livers on the bench. with a mean ischemia time of 7 ? 2 h, the perfusates contained on average 91 x 106 mononuclear cells (10c500 x 106). viability as determined by trypan blue exclusion showed that 98 ? 2% of the lpmc were vital. liver lymphocytes are known to express higher levels of lfa-1 than lymphocytes in peripheral blood (25, 26). flow cytometric characterization of lpmc and pbmc showed that within the perfusates, a significantly greater proportion of lymphocytes had high lfa-1 expression (fig. 1). the cd4 to cd8 ratio in perfusates was 1:2.4 (n = 22 human liver grafts) and was significantly distinct from the 2:1 ratio in blood from controls (n = 14; p < 0.001). these findings indicate that leukocytes present in perfusates are predominantly liver derived and not derived from residual blood.
figure 1. high lfa-1 expression on cd4+ t cells from perfusates. a, assessment of lfa-1 expression on cd4+ t cells in lpmc and pbmc. representative histogram showing higher lfa-1 (cd11a) expression on cd4+ t cells in lpmc compared with cd4+ t cells in pbmc. b, the increased proportion of lfa-1high cd4+ t cells in perfusates suggests that these cells are of liver origin and not derived from residual donor blood. in perfusates (n = 9) on average, 81% of cd4+ t cells are lfa-1high compared with 28% in pbmc of healthy controls (n = 10). data shown are mean percentages + sem.
lpmc are enriched for cd4+cd25+ctla4+ and cd4+cd25+foxp3Allosuppressive Donor CD+CD+ Regulatory T Cells Detach from the Graft论文+ t cells
to determine the presence of t cells with regulatory phenotype, cd25, ctla-4, and foxp3 expression within cd3+cd4+ t cells was assessed by flow cytometry. fig. 2a is a facs profile of cd3+cd4+cd25+ctla4+ cells of lpmc. as shown in fig. 2b, lpmc contained a median of 4.9% cd4+cd25+ctla4+ t cells (1.8c12.1%), significantly higher compared with 2.2% in pbmc of healthy controls (1.1c3.1%). a subset of perfusates was analyzed by staining for foxp3 expression in lpmc and pbmc (fig. 3a). comparison of perfusate and peripheral blood revealed increased proportions of cd4+cd25+foxp3+ t cells in perfusates (fig. 3b). rt-pcr confirmed the presence of foxp3+ t cells by analysis of foxp3 transcript levels in perfusates (data not shown). analysis of cd127, the il-7r, showed that the majority of perfusate foxp3+ cells were negative for cd127 identical with foxp3+ cells in blood (fig. 3, c and d). in sum, these data indicate that a considerable proportion of th cells that detach from the liver during perfusion have a regulatory phenotype.
figure 2. lpmc cd4+ t cells contain a higher cd25+ctla4+ cell fraction compared with pbmc. a, facs profile of cd3+cd4+cd25+ctla4+ cells in lpmc. within the cd3+cd4+ t cell population, the proportion of cells expressing membrane cd25 and intracellular ctla-4 was determined. representative dot plot from liver perfusate shows gated isotype-matched control stainings. b, increased proportion of cd4+cd25+ctla4+ t cells in perfusates (n = 22) () compared with cells in peripheral blood (n = 14) (). median percentage of cd25+ctla4+ cells (horizontal bar) within cd3+cd4+ t cells is 4.9% (1.8c12.1%) in lpmc compared with 2.2% (1.1c3.1%) in pbmc.
figure 3. increased percentage of cd4+cd25+foxp3+ cells in lpmc compared with pbmc. assessment of cd4+cd25+foxp3+ t cells in perfusate and pbmc. within the cd3+cd4+ t cell population, the proportion of cells expressing cd25 and foxp3 was determined. a, dot plot analysis of perfusate by staining shows the percentage of lpmc and pbmc for each quadrant. b, increased cd25+foxp3+ cells in total cd4+ t cells in perfusate compared with pbmc. almost all cd4+foxp3+ were positive for cd25. c, dot plot showing cd127 expression on perfusate cd4+ cells. d, the majority of perfusate cd4+foxp3+ cells (n = 4) are cd127-negative compared with the expression in blood cd4+foxp3+ cells (n = 6). the difference in the percentage of cd127-negative was not significant (n.s.) comparable to cells in control blood.
perfusate cd4+cd25+ t cells inhibit proliferation and ifn- production of donor and recipient responder t cells
to test the suppressive activity of cd4+cd25+ t cells in lpmc, we performed mlr analysis using cfse fluorescent-labeled responder t cells. first, we tested the suppressive activity of cd4+cd25+ cells in the autologous setting. donor cd4+cd25c responder cells (1 x 105) from lpmc were mixed with two different concentrations of donor cd4+cd25+ t cells leading to a dose-dependent inhibition of proliferation as shown in fig. 4. also, the autologous suppressive activity of recipient cd4+cd25+ t cells from pmbc was demonstrated. notably, the proliferation of perfusate cd4+cd25c t cells was less extensive compared with recipient blood responder t cells. however, the suppressive activity of perfusate cd4+cd25+ t cells was intact.
figure 4. perfusate cd4+cd25+ t cells inhibit proliferation of both recipient and donor responder t cells. a, representative mlr showing cfse staining of control recipient cd4+cd25c t cells (1 x 105) in the absence (left) and presence (right) of perfusate cd4+cd25+ t cells (3 x 104) upon stimulation with irradiated donor and recipient mononuclear cells. the number of cells in the daughter generations is significantly lowered in the presence of perfusate cd4+cd25+ t cells. b, effect of perfusate and recipient cd4+cd25+ t cells on proliferation of recipient and donor cd4+cd25c t cells. proliferation of recipient (1 x 105) () and perfusate (1 x 105) () responder cells was significantly inhibited by perfusate cd4+cd25+ t cells. *, p = 0.034 and **, p = 0.032, respectively. responder cells were stimulated with irradiated donor (5 x 104) and recipient mononuclear cells (5 x 104). as a positive control the suppressive activity of recipient cd4+cd25+ t cells () was also determined. ***, p = 0.003. significance was tested by analysis of the logarithmic transformation of the dependent variable with random intercept and random slope. data are the mean ? sem of n = 5 experiments.
next we tested the suppressive activity of perfusate cd4+cd25+ t cells in an allogeneic setting. as apcs, both irradiated pbmc (5 x 104) and lpmc (5 x 104) were used together to simultaneously activate recipient (responder) and donor (suppressor) t cells. both perfusate and recipient cd4+cd25+ cells showed no proliferation in response to allogeneic stimulation, indicating a state of anergy (data not shown). when cd4+cd25+ t cells from lpmc were added to recipient cd4+cd25c responder cells, we observed significant suppression of proliferation comparable to the inhibition observed in the autologous setting (fig. 4). consistently, when looking at the ifn- production in these mlr analyses, similar results were found showing dose-dependent inhibition by donor cd4+cd25+ t cells of donor and recipient ifn- production (fig. 5). the suppression of both proliferation and ifn- production suggests that cd4+cd25+ t cells from the liver are able to suppress the direct pathway alloresponse in vitro.
figure 5. inhibition of ifn- production by perfusate cd4+cd25+ t cells. cd4+cd25+ t cells inhibit ifn- production by effector cd4+cd25c t cells. data are the mean ? sem of the relative inhibition of ifn- production for three experiments, as determined by elisa. perfusate cd4+cd25+ t cells inhibit ifn- production of recipient responder cd4+cd25c t cells () (p = 0.03) and perfusate responder cd4+cd25c t cells () (p = 0.004). recipient cd4+cd25+ t cells () inhibit ifn- production of recipient responder cd4+cd25c t cells (p = 0.07).
cd4+cd25+ctla4+ t cells detach from the liver graft after tx and circulate in recipients
to determine whether donor t cells with a regulatory phenotype can be detected in recipients after liver tx, we performed flow cytometric analysis of pbmc from hla-a2-negative recipients who received an hla-a2-positive liver graft. determination of hla-a2 class 1 allele has previously been shown to be useful to distinguish between donor and recipients cells (27, 28). a representative staining is shown in fig. 6a. in the first week after tx, 3.1 ? 1.0% (?sem) of the total cd4+ t cells and 2.5 ? 1.0% (?sem) of the cd4+cd25+ctla4+ cells were found to be of donor origin (fig. 6b). these percentages of donor cells increased initially and gradually declined at 1 and 6 mo after tx. similar results were found when analyzing the percentage of hla-a2-positive cells within the cd4+cd25+foxp3+ fraction (n = 4; data not shown). the proportion of cd25+ctla4+ cells within cd4+ cells was significantly higher in donor-derived than in recipient cells (fig. 6c). there was also a difference at 6 mo, although the overall percentage of cd4+cd25+ctla4+ cells had declined at this time point. these data indicate that donor t cells with a regulatory phenotype migrate from the liver graft into the circulation of recipients.
figure 6. donor cd4+cd25+ctla4+ t cells circulate in recipients after liver tx. a, dot plots represent donor hla-a2-positive cells in recipient blood 1 wk post-tx. pre-tx recipient pbmc and donor spleen cells serve as negative and positive control, respectively. the percentage of cd25 and ctla-4 expressing hla-a2-positive and hla-a2-negative cd4+ cells is shown in each quadrant. b, proportion of hla-a2-positive cells within the cd4+ cells and within cd4+cd25+ctla4+ cells is highest at 1 wk (n = 6) and gradually decreases at 1 mo (n = 4) and 6 mo (n = 4) after tx. c, at 1 wk and 6 mo after tx, the proportion of donor cd4+cd25+ctla4+ cells within the donor cd4+ cells was increased compared with the proportions of cd4+cd25+ctla4+ cells within recipient cd4+ cells. *, p < 0.05. both recipient and donor cd4+cd25+ctla4+ cells decrease at 1 and 6 mo after tx, although not reaching statistical significance due to small sample size.
the unique immunological properties of the liver have partly been attributed to the resident leukocyte population. following liver tx, donor leukocytes present in the graft are transferred and persist in recipients, a condition referred to as chimerism. in this study we aimed to investigate the presence of tregs in liver graft perfusion solution and in recipient blood after tx. within perfusate cd3+cd4+ t cells an increased proportion of cd25+ctla4+ and cd25+foxp3+ t cells were found as compared with cells found in peripheral blood of healthy controls (figs. 2 and 3). foxp3 mrna expression in lpmc confirmed the presence of treg (data not shown). leukocytes in recipient circulation were shown to contain substantial numbers of donor cd4+cd25+ctla4+ t cells indicative of a regulatory signature (fig. 6). the functionality of donor treg was studied in mlr, showing inhibition of proliferation of self- and recipient-responder cd4+cd25c t cells upon stimulation with donor and recipient mononuclear cells (fig. 4). furthermore, the cytokine production was significantly inhibited, confirming the suppressive activity of cd4+cd25+ t cells within the lpmc (fig. 5).
the number of mononuclear cells obtained from perfusates showed a wide range, but did not correlate with ischemia time or perfusate volume (data not shown). immunophenotypic characterization of lpmc shows clear differences when compared with pbmc, yet comparable with leukocyte subsets obtained from liver tissue (23, 24). our results show significantly lower proportions of cd4+ t cells and higher proportions of cd8+ within total lpmc t cell population compared with proportions found in peripheral blood. previously, it was shown that liver-infiltrating t cells have high expression levels of the 2 integrin lfa-1 (22, 25). therefore we determined the expression of lfa-1 -chain (cd11a), which is expressed exclusively on leukocytes and is involved in migration into tissues. the cd4+ t cells in perfusates have a strong lfa-1 expression, which is significantly higher compared with cd4+ t cells in pbmc (fig. 1). the strong adhesion molecule expression and the reversed cd4 to cd8 ratio indicate that these cells originate from the liver tissue and have a preactivated phenotype (22, 29).
within the cd4+ t cell population obtained from perfusates, a considerable proportion of t cells with a regulatory phenotype was observed. tregs characterized by cd4, cd25, ctla-4, and foxp3 expression were significantly increased compared with peripheral blood levels in healthy controls (figs. 2 and 3). recently, the absence of the il-7r cd127 expression has been suggested as a discriminating factor of foxp3+ treg (30, 31). our analysis showed that the majority of liver-derived foxp3+ cells were cd127-negative, which was comparable to cells in control blood (fig. 3d). to our knowledge, this study is the first to show the presence of cd4+cd25+foxp3+ t cells as passenger leukocytes in liver grafts. following liver tx, a substantial proportion of circulating leukocytes is of donor origin (28) and even higher in the case of graft-vs-host disease (32). in the first week after tx, we have shown that up to 5% of the total cd4+cd25+ctla4+ t cell population were of donor origin (fig. 6b), equivalent to 5c15 x 106 cells. the proportion of cd4+ cells that expressed cd25 and ctla-4 was higher in donor cells than in recipient cells at 1 and 6 mo after tx (fig. 6c). both donor and recipient cd4+cd25+ctla4+ cells decreased in time after tx. this general drop of cd4+cd25+ctla4+ tregs was consistent with our previous studies (33, 34).
katz et al. (35) have shown that hepatic cd4+ t cells are functionally suppressed by environmental factors, which may explain the low proliferative capacity of cd4+cd25c responder cells from lpmc in our study. as we have shown that a relatively increased proportion of cd4+ t cells present in the liver have a regulatory phenotype, one might postulate that hepatic tregs are involved in mediating suppression of responses to ags presented in the liver. in the context of tx, these hepatic tregs may suppress not only alloantigen-specific recipient t cells but also dc and thereby inhibit the immune response against the graft. in our previous study we have shown that hepatic dc have an immature phenotype and produce high amounts of il-10 (24). experimental evidence suggests that immature dc can mediate tolerance, presumably by the induction of tregs (36, 37). furthermore, in vitro, immature dc can induce alloantigen-reactive tregs (38). liver-derived dc are able to down-regulate immune responses and stimulate t cells to produce il-10 and il-4 (39, 40, 41). however, tregs limit the ability of dc to stimulate t cells and enhance the ability of dc to induce anergy concomitant with an increase in ctla-4 expression (42, 43, 44). this way, a bidirectional interaction may occur between liver t cells and dc, which may explain the unique hepatic microenvironment that is known to promote tolerance.
in this study we have shown that donor cd4+cd25+ t cells originating from the liver graft are able to suppress responder t cells from both recipient and donor. this observation suggests that tregs canAllosuppressive Donor CD+CD+ Regulatory T Cells Detach from the Graft论文 suppress across a mhc barrier. this work is consistent with previous findings showing that tregs suppress ag nonspecifically once activated through their tcr (45). furthermore, recently it was shown that allogeneic treg can inhibit mhc-disparate responder t cells (46). these data indicate that chimerism of donor treg may contribute to suppression of the direct pathway alloresponse that is the dominant ag presentation pathway driving rejection early after tx. the immunological relevance of donor leukocytes for allograft acceptance is particularly evident during the early phase after tx (3). as we have shown that cd4+cd25+ t cells are functional in suppressing responder cells, one might postulate that these cells participate in the silencing of donor reactive t cells. these findings fit with the possibility that chimerism plays a key role in acquired tx tolerance.
donor leukocyte migration and chimerism are associated with transplant tolerance in both the clinical and experimental setting (1, 2, 47). depletion of donor leukocytes or failure to develop chimerism was shown to result in prompt rejection, but chimerism by itself is not sufficient to prevent rejection. in clinical tx, the beneficial effect of donor leukocytes was clearly shown by pre-tx blood transfusions leading to enhanced graft survival (48, 49). it is evident that donor leukocytes are important during the first weeks post-tx in which maximal donor migration and interaction with host leukocytes occur. the exact mechanism in achieving tolerance through chimerism, however, has not been elucidated. several mechanisms have been proposed in this process including clonal exhaustion or deletion, t cell anergy, and active suppression (8, 10). although the cell interactions are indisputably complex, tolerance to skin transplants following bone marrow tx was cd4+cd25+ treg-dependent mostly early after tx (19). conversely, in bone marrow tx, an important role for cd4+ tregs has recently been identified in the induction of chimerism-associated tolerance, which was cd25-independent but ctla-4-dependent (20). whether graft-derived donor tregs influence the balance between the immunogenicity and tolerogenicity of organ allografts remains to be determined.
in conclusion, a substantial number of donor tregs detach from the liver graft during perfusion and continue to migrate into the recipient after tx. these donor tregs are functional in suppressing the direct pathway alloresponses in vitro and may therefore contribute to chimerism-associated tolerance early after liver tx.
we thank dr. g. kazemier, dr. t. c. k. tran, and dr. i. p. j. alwayn for help obtaining the perfusate solutions. we thank dr. b. hansen for performing statistical analysis, dr. r. van der molen for the gift of anti-hla-a2 ab, and i. e. dijke for technical assistance with cd127 facs analysis.
the authors have no financial conflict of interest.
starzl, t. e., a. j. demetris, n. murase, s. ildstad, c. ricordi, m. trucco. 1992. cell migration, chimerism, and graft acceptance. lancet 339: 1579-1582.
starzl, t. e., a. j. demetris, m. trucco, n. murase, c. ricordi, s. ildstad, h. ramos, s. todo, a. tzakis, j. j. fung, et al 1993. cell migration and chimerism after whole-organ transplantation: the basis of graft acceptance. hepatology 17: 1127-1152.
ko, s., a. deiwick, m. d. jager, a. dinkel, f. rohde, r. fischer, t. y. tsui, k. l. rittmann, k. wonigeit, h. j. schlitt. 1999. the functional relevance of passenger leukocytes and microchimerism for heart allograft acceptance in the rat. nat. med. 5: 1292-1297.
sun, j., g. w. mccaughan, n. d. gallagher, a. g. sheil, g. a. bishop. 1995. deletion of spontaneous rat liver allograft acceptance by donor irradiation. transplantation 60: 233-236.
sriwatanawongsa, v., h. s. davies, r. y. calne. 1995. the essential roles of parenchymal tissues and passenger leukocytes in the tolerance induced by liver grafting in rats. nat. med. 1: 428-432.
sun, j., a. g. sheil, c. wang, l. wang, k. rokahr, a. sharland, s. e. jung, l. li, g. w. mccaughan, g. a. bishop. 1996. tolerance to rat liver allografts: iv. acceptance depends on the quantity of donor tissue and on donor leukocytes. transplantation 62: 1725-1730.
anderson, c. c., p. matzinger. 2001. immunity or tolerance: opposite outcomes of microchimerism from skin grafts. nat. med. 7: 80-87.
kurtz, j., t. wekerle, m. sykes. 2004. tolerance in mixed chimerism: a role for regulatory cells?. trends immunol. 25: 518-523.
claas, f.. 2004. chimerism as a tool to induce clinical transplantation tolerance. curr. opin. immunol. 16: 578-583.
starzl, t. e.. 2004. chimerism and tolerance in transplantation. proc. natl. acad. sci. usa 101: (suppl. 2):14607-14614.
marcos, a., f. lakkis, t. e. starzl. 2006. tolerance for organ recipients: a clash of paradigms. liver transpl. 12: 1448-1451.
jonsson, j. r., p. g. hogan, r. thomas, c. steadman, a. d. clouston, g. a. balderson, s. v. lynch, r. w. strong, e. e. powell. 1997. peripheral blood chimerism following human liver transplantation. hepatology 25: 1233-1236.
van maurik, a., m. herber, k. j. wood, n. d. jones. 2002. cutting edge: cd4+cd25+ alloantigen-specific immunoregulatory cells that can prevent cd8+ t cell-mediated graft rejection: implications for anti-cd154 immunotherapy. j. immunol. 169: 5401-5404.
wood, k. j., s. sakaguchi. 2003. regulatory t cells in transplantation tolerance. nat. rev. immunol. 3: 199-210.
graca, l., a. le moine, c. y. lin, p. j. fairchild, s. p. cobbold, h. waldmann. 2004. donor-specific transplantation tolerance: the paradoxical behavior of cd4+cd25+ t cells. proc. natl. acad. sci. usa 101: 10122-10126.
lee, m. k., iv, d. j. moore, b. p. jarrett, m. m. lian, s. deng, x. huang, j. w. markmann, m. chiaccio, c. f. barker, a. j. caton, j. f. markmann. 2004. promotion of allograft survival by cd4+cd25+ regulatory t cells: evidence for in vivo inhibition of effector cell proliferation. j. immunol. 172: 6539-6544.
kingsley, c. i., m. karim, a. r. bushell, k. j. wood. 2002. cd25+cd4+ regulatory t cells prevent graft rejection: ctla-4- and il-10-dependent immunoregulation of alloresponses. j. immunol. 168: 1080-1086.
vasu, c., b. s. prabhakar, m. j. holterman. 2004. targeted ctla-4 engagement induces cd4+cd25+ctla-4high t regulatory cells with target (allo)antigen specificity. j. immunol. 173: 2866-2876.
bigenzahn, s., p. blaha, z. koporc, i. pree, e. selzer, h. bergmeister, f. wrba, c. heusser, k. wagner, f. muehlbacher, t. wekerle. 2005. the role of non-deletional tolerance mechanisms in a murine model of mixed chimerism with costimulation blockade. am. j. transplant. 5: 1237-1247.
fehr, t., y. takeuchi, j. kurtz, t. wekerle, m. sykes. 2005. early regulation of cd8 t cell alloreactivity by cd4+cd25c t cells in recipients of anti-cd154 antibody and allogeneic bmt is followed by rapid peripheral deletion of donor-reactive cd8+ t cells, precluding a role for sustained regulation. eur. j. immunol. 35: 2679-2690.
domenig, c., a. sanchez-fueyo, j. kurtz, s. p. alexopoulos, c. mariat, m. sykes, t. b. strom, x. x. zheng. 2005. roles of deletion and regulation in creating mixed chimerism and allograft tolerance using a nonlymphoablative irradiation-free protocol. j. immunol. 175: 51-60.
schlitt, h. j., g. raddatz, g. steinhoff, k. wonigeit, r. pichlmayr. 1993. passenger lymphocytes in human liver allografts and their potential role after transplantation. transplantation 56: 951-955.
jonsson, j. r., p. g. hogan, g. a. balderson, l. l. ooi, s. v. lynch, r. w. strong, e. e. powell. 1997. human liver transplant perfusate: an abundant source of donor liver-associated leukocytes. hepatology 26: 1111-1114.
bosma, b. m., h. j. metselaar, s. mancham, p. p. boor, j. g. kusters, g. kazemier, h. w. tilanus, e. j. kuipers, j. kwekkeboom. 2006. characterization of human liver dendritic cells in liver grafts and perfusates. liver transpl. 12: 384-393.
steinhoff, g., m. behrend, k. wonigeit. 1990. expression of adhesion molecules on lymphocytes/monocytes and hepatocytes in human liver grafts. hum. immunol. 28: 123-127.
heydtmann, m., d. hardie, p. l. shields, j. faint, c. d. buckley, j. j. campbell, m. salmon, d. h. adams. 2006. detailed analysis of intrahepatic cd8 t cells in the normal and hepatitis c-infected liver reveals differences in specific populations of memory cells with distinct homing phenotypes. j. immunol. 177: 729-738.
schlitt, h. j., h. kanehiro, g. raddatz, g. steinhoff, n. richter, b. nashan, b. ringe, k. wonigeit, r. pichlmayr. 1993. persistence of donor lymphocytes in liver allograft recipients. transplantation 56: 1001-1007.
heerwagen, c., m. schuster, a. bornscheurer, l. pape, e. kirchner, h. j. schlitt, b. luettig, j. westermann. 2001. rapid exchange of large numbers of donor- and host leukocytes after human liver transplantation. transpl. int. 14: 240-247.
sato, t., a. habtezion, a. beilhack, s. schulz, e. butcher, h. thorlacius. 2006. short-term homing assay reveals a critical role for lymphocyte function-associated antigen-1 in the hepatic recruitment of lymphocytes in graft-versus-host disease. j. hepatol. 44: 1132-1140.
liu, w., a. l. putnam, z. xu-yu, g. l. szot, m. r. lee, s. zhu, p. a. gottlieb, p. kapranov, t. r. gingeras, b. fazekas de st groth, et al 2006. cd127 expression inversely correlates with foxp3 and suppressive function of human cd4+ t reg cells. j. exp. med. 203: 1701-1711.
seddiki, n., b. santner-nanan, j. martinson, j. zaunders, s. sasson, a. landay, m. solomon, w. selby, s. i. alexander, r. nanan, et al 2006. expression of interleukin (il)-2 and il-7 receptors discriminates between human regulatory and activated t cells. j. exp. med. 203: 1693-1700.
domiati-saad, r., g. b. klintmalm, g. netto, e. d. agura, s. chinnakotla, d. m. smith. 2005. acute graft versus host disease after liver transplantation: patterns of lymphocyte chimerism. am. j. transplant. 5: 2968-2973.
demirkiran, a., a. kok, j. kwekkeboom, h. j. metselaar, h. w. tilanus, l. j. van der laan. 2005. decrease of cd4+cd25+ t cells in peripheral blood after liver transplantation: association with immunosuppression. transplant proc. 37: 1194-1196.
demirkiran, a., a. kok, j. kwekkeboom, j. g. kusters, h. j. metselaar, h. w. tilanus, l. j. van der laan. 2006. low circulating regulatory t-cell levels after acute rejection in liver transplantation. liver transpl. 12: 277-284.
katz, s. c., v. g. pillarisetty, j. i. bleier, t. p. kingham, u. i. chaudhry, a. b. shah, r. p. dematteo. 2005. conventional liver cd4 t cells are functionally distinct and suppressed by environmental factors. hepatology 42: 293-300.
fairchild, p. j., h. waldmann. 2000. dendritic cells and prospects for transplantation tolerance. curr. opin. immunol. 12: 528-535.
steinman, r. m., s. turley, i. mellman, k. inaba. 2000. the induction of tolerance by dendritic cells that have captured apoptotic cells. j. exp. med. 191: 411-416.
jonuleit, h., e. schmitt, g. schuler, j. knop, a. h. enk. 2000. induction of interleukin 10-producing, nonproliferating cd4+ t cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. j. exp. med. 192: 1213-1222.
thomson, a. w., t. takayama. 1999. dendritic cells and the outcome of organ transplantation: a contemporary view. transplant proc. 31: 2738-2739.
thomson, a. w., l. lu. 1999. are dendritic cells the key to liver transplant tolerance?. immunol. today 20: 27-32.
goddard, s., j. youster, e. morgan, d. h. adams. 2004. interleukin-10 secretion differentiates dendritic cells from human liver and skin. am. j. pathol. 164: 511-519.
min, w. p., d. zhou, t. e. ichim, g. h. strejan, x. xia, j. yang, x. huang, b. garcia, d. white, p. dutartre, et al 2003. inhibitory feedback loop between tolerogenic dendritic cells and regulatory t cells in transplant tolerance. j. immunol. 170: 1304-1312.
steinbrink, k., e. graulich, s. kubsch, j. knop, a. h. enk. 2002. cd4+ and cd8+ anergic t cells induced by interleukin-10-treated human dendritic cells display antigen-specific suppressor activity. blood 99: 2468-2476.
misra, n., j. bayry, s. lacroix-desmazes, m. d. kazatchkine, s. v. kaveri. 2004. cutting edge: human cd4+cd25+ t cells restrain the maturation and antigen-presenting function of dendritic cells. j. immunol. 172: 4676-4680.
thornton, a. m., e. m. shevach. 2000. suppressor effector function of cd4+cd25+ immunoregulatory t cells is antigen nonspecific. j. immunol. 164: 183-190.
adeegbe, d., a. l. bayer, r. b. levy, t. r. malek. 2006. cutting edge: allogeneic cd4+cd25+foxp3+ t regulatory cells suppress autoimmunity while establishing transplantation tolerance. j. immunol. 176: 7149-7153.
kawai, t., a. b. cosimi, r. b. colvin, j. powelson, j. eason, t. kozlowski, m. sykes, r. monroy, m. tanaka, d. h. sachs. 1995. mixed allogeneic chimerism and renal allograft tolerance in cynomolgus monkeys. transplantation 59: 256-262.