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*
Department of Surgery and Multiorgan Transplant Program, Toronto Hospital Research Institute, University of Toronto, Toronto, Ontario, Canada; and
Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada
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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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Genetic modification of DC with genes encoding immunoregulatory molecules is an alternative approach for artificial generation of tolerogenic DC. Indeed, recent reports suggest that transfection of DC with IL-10 and TGF-ß can increase their tolerogenic potential (6, 7). Several attributes make DC ideal vehicles for the delivery of such molecules. They are potent activators of naive T cells, a function related to their Ag-processing capacity and to high levels of expression of MHC and costimulatory molecules. In addition, they have unique migratory capability, enabling them to move from peripheral tissues to secondary lymphoid organs, where they interact with T and B cells (8, 9). The cognate recognition of DC and T cells provides the theoretic opportunity of these immunomodulatory molecules to influence the immune response in an Ag-specific manner.
One molecule that may enhance the tolerance-inducing capacity of DC is Fas ligand (FasL), a type II integral membrane protein that belongs to the TNF superfamily (10). Engagement of Fas by FasL initiates a signaling cascade that leads to apoptotic cell death of Fas-bearing cells. Apoptosis induced by Fas/FasL interactions is thought to play a pivotal role in the immune system, regulating both peripheral T cell homeostasis and lymphocyte-mediated cytotoxicity. FasL is expressed in immunoprivileged organs, including the eye and testis, where it has been proposed to contribute to their tolerogenic milieu and paucity of infiltrating inflammatory cells (11, 12, 13). There is evidence that FasL constitutively expressed on splenic DC and bone marrow-derived DC may be involved in the killing of activated CD4+ T cells (14, 15). More recently, tolerance induced by infusion of donor bone marrow cells was shown to be dependent on the expression of FasL on the infused cells (16). In the studies described below, we investigate the immunomodulatory effect of DC transduced to express high levels of FasL in vitro and in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male C57BL/6 (H-2b), BALB/c (H-2d), and C3H (H-2k) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6-lpr/lpr mice were purchased originally from The Jackson Laboratory and bred in our animal facility. All mice were used at 8–12 wk of age.
Generation of bone marrow-derived DC
DC were generated from bone marrow progenitor cells, as described by Inaba et al. (17) and modified by Suri et al.4 Briefly, bone marrow cells were flushed from femurs and tibias of C57BL/6 mice, washed, and cultured in 6-well plates (2 x 106/ml) in 4 ml RPMI 1640 containing rGM-CSF (10 ng/ml; Peprotech, Rocky Hill, NJ) and mouse rIL-4 (10 ng/ml; Peprotech). All media and additives were documented to be free of LPS contamination (18). Nonadherent granulocytes were removed after 48 h of culture, and fresh media added every 48 h. By day 4 to 6 of culture, proliferating clusters of cells with typical dendritic morphology were seen, and by day 7 to 9 more than 90% of the cells expressed the DC cell surface marker DEC-205. The proportion of cells staining for T (CD3) and B (B220) lymphocytes was consistently <3%.
Gene transfection
pBK-CMV phagemid vector (2 µg) containing full-length human FasL cDNA or empty control vector was incubated with 8 µl of Lipofectin (Life Technologies, Gaithersburg, MD) in a volume of 100 µl of PBS at room temperature for 45 min. This mixture was added to 7-day cultured DC in a final volume of 1 ml of serum-free medium. After 4-h incubation at 37°C with 5% CO2, the cells were washed and cultured in RPMI 1640 with 10% FCS for 48 h.
Flow cytometry
Phenotypic analysis of DC was performed at day 9 (2 days after
gene transfection) of culture using an EPICS XL-MCL Cell Analysis
System (Coulter, Miami, FL). The following mAbs were purchased from
Cedarlane Laboratories (Hornby, Ontario, Canada), unless otherwise
indicated, and used for staining cells as primary mAbs: anti-DEC205
(clone NLDC-145), anti-mouse H-2b, anti mouse
I-Eb, and anti-mouse CD40 (PharMingen, San
Diego, CA). The secondary mAbs used were FITC-conjugated anti-rat
IgG2a (Caltag Laboratories) or FITC-conjugated anti-mouse IgG2a
(PharMingen). The following mAbs, purchased from PharMingen, were used
directly as FITC-conjugated mAb: anti-mouse CD80 (B7-1),
anti-mouse CD86 (B7-2), anti-mouse CD3
, anti-mouse CD4,
anti-mouse CD8, anti-mouse Mac1, and anti-mouse B220.
FasL-transfected and control DC were stained with anti-human FasL
mAb (MBL, Nagoya, Japan) or isotype control, followed by secondary
PE-conjugated anti-hamster IgG (Cedarlane Laboratories)
RT-PCR
Total RNA was extracted from DC (1 x 107) 48 h after transfection with FasL or the empty control vector, with TRIzol reagent (Life Technologies), as per the manufacturer’s instructions. First strand cDNA was synthesized using an RNA PCR kit (Life Technologies) with the supplied oligo(dT)16 primer.
One microliter of the reverse-transcription reaction product was used for the subsequent PCR reaction. The sequence of the human FasL primers, which generated a 293-bp fragment of human FasL, was: sense, 5'-AATAGGCCACCCCAGTCCA-3'; antisense, 5'-CCCCTCCATCATCACCAGA-3. The sequence of the mouse ß-actin primers was: sense, 5'-AGGCATCCTGACCCTGAAGTAC-3'; antisense, 5'-TCTTCATGAGGTAGTCTGTCAG-3'. The samples were denaturated for 1 min at 94°C, annealed for 1 min at 53°C, and extended for 1 min at 72°C, for a total of 35 cycles. The PCR products were subjected to electrophoresis on 1.5% agarose gel containing ethidium bromide and visualized by UV illumination. ß-actin was used as an internal control for RNA integrity.
DNA fragmentation (JAM assay)
Jurkat cells (1 x 104) were labeled with 5 µCi/ml [3H]thymidine for 4 h at 37°C (19) and seeded in triplicate in U-bottom 96-well tissue culture plates as target cells. They were incubated with the indicated ratio of FasL- or control-transfected DC in a total volume of 200 µl/well for 18 h. Unfragmented high m.w. DNA was harvested onto glass fiber filters and counted in a Beckman scintillation counter. Data are expressed as percentage of DNA fragmentation: 100 x [(1 - cpm in experimental group)/(cpm of unstimulated targets alone)].
Mixed leukocyte reactions
Two days after gene transfection, FasL- or control-transfected DC (1 x 104) were treated with 50 µg/ml of mitomycin C at 37°C for 20 min, washed twice with RPMI, and seeded in triplicate in flat-bottom 96-well culture plates (Corning Glass, Corning, NY) for use as stimulator cells. Responder spleen cells (2 x 105/well) from BALB/c mice were added to the DC in a total volume of 200 µl of RPMI 1640 containing 10% FCS, 50 µM 2-ME, 1 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin, and cultured in a humidified atmosphere of 5% CO2 in air at 37°C. The cells were pulsed with 1 µCi of [3H]thymidine (Amersham, Arlington Heights, IL) at the indicated time points, cultured an additional 16 h, and collected onto glass fiber filters; [3H]thymidine incorporation was quantified using a Beckman scintillation counter. Results were expressed as the mean cpm of triplicate cultures.
For MLR using C57BL/6-lpr/lpr responder splenocytes, the stimulating DC were generated from BALB/c mice and transfected as described above.
Blockade of FasL
Soluble Fas:Fc fusion protein (Alexis, San Diego, CA), or control human IgG (Calbiochem, San Diego, CA) was added at the beginning of MLR and DNA fragmentation assays at the concentrations indicated, and as described by Desbarats et al. (20).
Flow-cytometric analysis for T cell apoptosis
Quantitative determination of T cell apoptosis was analyzed by flow cytometry, as described by Nicoletti et al. (21). Purified T cells were activated with Con A (5 µg/ml) for 72 h and collected over Ficoll-Hypaque. Viable blasts (5 x 106) were incubated with 5 x 105 FasL-transfected or control DC for 24 h. Cell suspensions were centrifuged at 200 x g for 10 min, gently resuspended in 1 ml of hypotonic fluorochrome solution (50 µg/ml propidium iodide, 3.4 mM sodium citrate, 1 mM Tris, 0.1 mM EDTA, 0.1% Triton X-100), and stored in the dark for 3–4 h before being analyzed by flow cytometry. The apoptotic cells were quantified as the percentage of cells with subdiploid DNA.
Donor-specific hyporesponsiveness induced by allogeneic DC injection
FasL- or empty control vector-transfected BALB/c DC (2 x 106) were injected i.p. into groups of nine C57BL/6 and four C57BL/6-lpr/lpr mice at 3-day intervals for a total of six injections, as per Zhang et al. (22). Mice were sacrificed 3 days after the last injection, and MLR cultures were initiated with fresh mitomycin C-treated BALB/c spleen stimulator cells, as described above. Paraffin sections of Formalin-fixed liver and spleen biopsies from these animals were stained with hematoxylin and eosin for histologic evaluation.
Heterotopic heart transplantation with DC pretreatment
FasL- or empty control vector-transfected BALB/c DC (2 x 106) were injected i.p. into groups of six C57BL/6 at 3-day intervals for a total of six injections. Within 3 days of the last injection, vascualized heterotopic heart transplants from BALB/c mice were performed and monitored daily, as described (23). Rejection was defined by the cessation of heartbeat.
Statistical analysis
Continuous variables were compared with Student t tests. Cardiac graft survival curves were calculated by the Kaplan-Meier method, with differences between groups compared by the log-rank test. A p value <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DC were propagated from bone marrow cells cultured with GM-CSF and IL-4, as described in Materials and Methods. By the seventh day of culture, cells with characteristic DC morphology and immunophenotype (DEC-205+, MHC class II+, CD40+, CD80+, CD862+, Mac1low, B220-, CD3-, CD4-, CD8-) were observed.
To optimize gene delivery into DC by lipofection, we first used the
Escherichia coli ß-galactosidase gene under the control of
the CMV immediate promoter as a reporter system. The optimal DNA
(µg):Lipofectin (µl) ratio was found to be 1:4 using an incubation
time of 4 h. With these conditions, the transfection efficacy was
50–70%, and cell viability was more than 90% (data not shown).
Transfection of DC with the FasL vector construct using the same
conditions resulted in high levels of FasL gene expression, but not
with the empty control vector, as determined by RT-PCR and
flow-cytometric analysis (Fig. 1
).
Forty-eight hours after transfection, 
50% of the DC stained
positive for FasL (range, 29% to 68% in 20 independent experiments).
Transfection with either FasL or the control vector did not adversely
affect cell viability or the expression levels of cell surface
molecules (Fig. 2).
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A). DC
transfected with FasL induced high levels of DNA fragmentation as
compared with those transfected with the control vector. Moreover, we
found that the addition of blocking Fas-Fc effectively inhibited DNA
fragmentation. This inhibition was dose dependent and specific, as it
was unaffected by the addition of control Ig (Fig. 3B).
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To assess the functional activity of transfected DC in stimulating
allogeneic T cell responses, MLR reactions were performed using FasL-
or control-transfected DC. Allogeneic T cell proliferation was
significantly decreased when incubated with FasL-DC, but not with the
control DC (Fig. 4A). The
stimulatory capacity of FasL-DC could be restored in the presence of
soluble Fas-Fc, but not control Ig, indicating that inhibition of
allogeneic MLR by FasL-DC was specific to FasL (Fig. 4B).
Furthermore, these results confirmed that DC transfected with FasL were
viable and capable of presenting alloantigen.
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To further establish the functional relevance of Fas:FasL
interactions in the inhibition of the MLR response by FasL-DC, the MLR
studies were repeated with lymphocytes from C57BL/6-lpr/lpr
mice, which do not express Fas. In these experiments, DC were
propagated from BALB/c bone marrow cells and transfected as described
above. FasL-DC inhibited the proliferative response of wild-type
lymphocytes, whereas there was no inhibition of
C57BL/6-lpr/lpr lymphocytes (Fig. 5).
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To explore the mechanism by which FasL-DC inhibit MLR, we
determined their ability to induce apoptosis of activated T cells using
a quantitative fluorometric assay for hypodiploid DNA. Con A blasts
were incubated with FasL-transfected or control-transfected DC for
24 h. As shown in Fig. 6, DC
transfected with FasL induced significantly higher levels of apoptosis
than the control-transfected cells.
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To determine whether Ag-specific inhibition of alloreactivity would be seen after pretreatment of naive mice with FasL-DC, C57BL/6 or C57BL/6-lpr/lpr mice received i.p. injections of 2 x 106 BALB/c-derived DC (FasL or control vector transfected) at 3-day intervals for a total of six injections. Three days after the last injection, all mice were sacrificed and MLR cultures were initiated using spleen cells from all individuals stimulated with BALB/c (allogeneic) or C3H (third-party) mitomycin-treated spleen stimulator cells. Control cultures used cells from nonimmunized (no DC treatment) mice.
FasL-DC treatment induced allospecific hyporesponsiveness during
restimulation with fresh stimulators in vitro, while splenocytes from
mice treated with control DC showed normal secondary responsiveness
(upper panel of Fig. 7). This
suppression of secondary proliferative responses by FasL-DC was
abrogated in lpr mice (lower panel of
Fig. 7).
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The capacity of FasL-DC to induce alloantigen-specific
hyporesponsiveness in vivo suggested that these cells might prolong
allograft survival. We tested this possibility with a vascularized
heterotopic cardiac transplant model. Groups of five to six C57BL/6
mice were pretreated with i.p. injections of 2 x
106 BALB/c control- and FasL-DC at 3-day
intervals for a total of six injections. As shown in Fig. 8, mean graft survival was significantly
longer in mice pretreated with FasL-DC as compared with both untreated
controls and those pretreated with control-DC (20 ± 4 vs 10
± 2 and 9 ± 3 days, respectively; p = 0.01 by
log-rank test).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A promising strategy for inducing tolerance to alloantigens is infusion
of tolerogenic DC. Previous studies have shown that immature DC, which
characteristically express low levels of costimulatory molecules such
as CD80 and CD86, can promote the development of donor-specific
tolerance and prolong cardiac and islet allograft survival (24, 25). In vitro growth conditions can be manipulated to enhance
the generation of immature DC from bone marrow cells (26, 27); however, subsequent maturation in vivo may limit their
tolerogenic potential (25). Genetic engineering of DC with
genes encoding immunoregulatory molecules provides an alternative
method of generating tolerogenic DC that might be more effective. The
feasibility of this approach is supported by recent studies showing
that DC can be genetically modified using retroviral and adenoviral
vectors to express model tumor Ags that promote both protective and
antitumor immunity (28, 29, 30), and cytokines that augment
(IFN-
, IL-12) and inhibit (IL-10, TGF-ß) immune responses
(6, 7, 31). Other molecules that could potentially enhance
the capacity of DC to promote tolerance include CTLA4-Ig, which would
block CD80 and CD86 costimulatory pathways, and OX-2, which we and
others have recently shown down-regulates T cell responses through a
unique costimulatory pathway (32, 33).
Apoptosis induced by Fas/FasL interactions is one mechanism implicated in peripheral T cell tolerance. Enhanced or elevated expression of FasL on specific tissues or cells by a transgene technique is being extensively applied for gene therapy of tumors (34) (35), rheumatoid arthritis (36), autoimmune diseases (37, 38), and regulation of rejection in transplantation (39, 40, 41). Thus, significant prolongation of allogeneic grafts has been achieved by directly transducing FasL gene into donor tissue or organs before transplantation (39, 40), or by cotransplanting FasL-transfected carrier cells (41). The studies we report suggest that DC transfected with FasL are capable of down-regulating T cell responses. This modulating effect appears to function by inducing T cell apoptosis via a Fas/FasL pathway, because inhibition was blocked by a Fas:Fc fusion protein, and failed to occur in lymphocytes from C57B6-lpr/lpr Fas mutant mice. Furthermore, we show that systemic administration of these cells not only inhibits donor-specific alloresponsiveness, but also prolongs cardiac allograft survival.
Our results are consistent with those of Zhang et al., who recently showed that a FasL-transfected macrophage cell line was capable of inducing allogeneic T cell hyporesponsiveness in vivo (22). They provide evidence with a CD8+ transgenic model that FasL-transfected macrophages are capable of inducing rapid and profound clonal deletion of Ag-specific T cells (22). It is interesting to consider the possibility that the peritoneal macrophage cell line used these studies, which express MAC1, F4/80, MHC class I and II, as well as significant amounts of B7 contained population(s) of DC (42). This would explain their finding of fluorochrome-labeled cells in splenic T cell areas after i.v. injection, which is a property characteristic of DC (43). Furthermore, it would be valuable to investigate whether the clonal deletion suggested in their study might be due to the migration of Ag-specific clones out of the spleen (44)or down-regulation of CD8 itself (45). Another potential mechanism that might contribute to the hyporesponsiveness induced by FasL-DC is polarization of Th cells toward a Th2 phenotype, as Th1 cells are reported to be more sensitive to FasL-mediated apoptosis than Th2 cells (46). These issues are currently under investigation in our laboratory using highly purified subsets of FasL-DC in an allospecific transgenic model.
Similarly, Matsue et al. have recently reported the generation of killer DC from an immortalized DC line (47). The authors show that peptide-pulsed FasL-expressing DC are capable of inducing Ag-specific T cell hyporesponsiveness in delayed-type hypersensitivity and contact hypersensitivity responses both prophylactically and therapeutically. The homing capacity and mechanism of action of these cells in vivo were not addressed, however. Interestingly, these killer DC were unable to block the induction of immune responses to alloantigen or Ab responses to nominal Ag, suggesting that there may be important differences between these cells and those used in our and Zhang’s (22) study.
Although our study indicates that FasL-DC can prolong survival of vascularized cardiac allografts, all grafts ultimately failed from rejection, suggesting that complete (or lasting) depletion of alloreactive cells did not occur. These results are in agreement with those of Matsue et al., who showed that hyporesponsiveness to dinitrofluorobenzene (DNFB) following treatment with a DNFB-pulsed FasL-DC clone was temporary and could be reversed with subsequent DNFB resensitization (47). Ongoing studies in our laboratory are being performed to determine whether the duration of allograft survival can be extended.
Earlier reports have shown that systemic administration of anti-Fas Abs and FasL-expressing viruses to mice causes massive hepatocyte apoptosis and liver failure, which has been attributed to high levels of expression of Fas on hepatocytes (48, 49). FasL also has proinflammatory properties mediated by recruitment and activation of neutrophils (50, 51). In our studies, however, treatment of mice with FasL-DC was remarkably well tolerated. Furthermore, histologic examination of livers from both the wild-type and lpr/lpr-treated mice showed no evidence of hepatitis or hepatocyte apoptosis (data no shown). Specific homing patterns of DC to secondary lymphoid organs may account for the lack of toxicity, and are currently being studied.
Recently, Matsue et al. reported that ligation of Fas on DC by FasL on T cells is capable of inducing DC apoptosis, and suggested that this may be one mechanism by which immune responses are normally terminated (52). Thus, one caveat of transfecting DC with FasL is that it might directly trigger DC apoptosis. However, the viability of DC after transfection in our studies was consistently greater than 90%, as determined by trypan blue exclusion and annexin V staining (data not shown). Whether FasL/Fas interactions induce DC apoptosis is most likely dependent on a variety of factors, including DC origin (e.g., spleen vs bone marrow), maturation stage, and expression levels of antiapoptotic proteins such as Bcl-2 and Bcl-xL (53, 54). The use of phenotypically mature DC (MHC IIhigh, DEC205+, CD40+, CD862+) in the present study, which express higher levels of Bcl-2 than immature DC, may account for their tolerability to FasL transfection. In addition, several ligand/receptor interactions have been shown to affect the outcome of FasL/Fas interaction. For example, DC survival signals expressed on T cells, such as TRANCE and CD40L, can prevent Fas-induced apoptosis (55, 56, 57).
Another potential limitation of using FasL-DC to inhibit immune responses is that the level of expression of functional Fas on naive T cells may be insufficient to trigger apoptosis. In fact, Nishimura et al. (58) have shown that murine T cells are resistant to anti-Fas mAb treatments. However, recent studies by Suda et al. (59) indicate that the membrane-bound form of FasL is capable of killing both fresh and in vitro activated peripheral blood T cells, whereas soluble FasL only kills the latter. The ability of FasL-DC to induce systemic T cell hyporesponsiveness in our study may be due, at least partly, to the use of the entire (membrane form) FasL molecule, which would be expected to provide a potent apoptotic signal.
A variety of gene delivery methods has been reported for transfecting DC, including viral vectors (7, 60, 61, 62), electroporation (22, 63), and gene guns (64). We have also used a replication-deficient adenoviral vector to transfect DC with FasL, but found that despite providing a high transfection efficiency, cell viability was significantly less than with the Lipofectin method used in the present study (unpublished observations). Whether this was due to direct viral injury or to the level of FasL expression is unclear. Other important advantages of liposomal gene transfer are the avoidance of potential biological hazards and antigenicity, which are associated with viral vectors.
In summary, the results of the present study suggest that transfection of DC with FasL may be a practical way to suppress allospecific immune responses in transplant recipients, and possibly for the treatment of autoimmune diseases. Furthermore, our results also highlight the potential of using DC genetically engineered to express other immunoregulatory genes. Currently, our efforts are directed toward optimizing gene delivery and expression in DC, and defining the conditions that maximize their ability to modulate in vivo immune responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mark S. Cattral, The Toronto Hospital, NU10-145, 621 University Avenue, Toronto, Ontario, Canada, M5G 2C4. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cell; DNFB, dinitrofluorobenzene; FasL, Fas ligand.
4 R. M. Suri, M. B. Lutz, A. L. J. Ogilvie, S. Robner, M. Nimi, N. Kukutsch, G. Schuler, and J. M. Austyn. 1999. Stably immature dendritic cells induce T cell unresponsiveness in vitro and prolong allograft survival in vivo. Submitted for publication.
Received for publication August 10, 1999. Accepted for publication October 19, 1999.
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M. Maksimow, M. Santanen, S. Jalkanen, and A. Hanninen Responding naive T cells differ in their sensitivity to Fas engagement: early death of many T cells is compensated by costimulation of surviving T cells Blood, May 15, 2003; 101(10): 4022 - 4028. [Abstract] [Full Text] [PDF]
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C. Guillot, S. Menoret, C. Guillonneau, C. Braudeau, M. G. Castro, P. Lowenstein, and I. Anegon Active suppression of allogeneic proliferative responses by dendritic cells after induction of long-term allograft survival by CTLA4Ig Blood, April 15, 2003; 101(8): 3325 - 3333. [Abstract] [Full Text] [PDF]
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S. Buonocore, F. Paulart, A. Le Moine, M. Braun, I. Salmon, S. Van Meirvenne, K. Thielemans, M. Goldman, and V. Flamand Dendritic cells overexpressing CD95 (Fas) ligand elicit vigorous allospecific T-cell responses in vivo Blood, February 15, 2003; 101(4): 1469 - 1476. [Abstract] [Full Text] [PDF]
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W.-P. Min, D. Zhou, T. E. Ichim, G. H. Strejan, X. Xia, J. Yang, X. Huang, B. Garcia, D. White, P. Dutartre, et al. Inhibitory Feedback Loop Between Tolerogenic Dendritic Cells and Regulatory T Cells in Transplant Tolerance J. Immunol., February 1, 2003; 170(3): 1304 - 1312. [Abstract] [Full Text] [PDF]
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T. Wolfe, C. Asseman, A. Hughes, H. Matsue, A. Takashima, and M. G. von Herrath Reduction of Antiviral CD8 Lymphocytes In Vivo with Dendritic Cells Expressing Fas Ligand--Increased Survival of Viral (Lymphocytic Choriomeningitis Virus) Central Nervous System Infection J. Immunol., November 1, 2002; 169(9): 4867 - 4872. [Abstract] [Full Text] [PDF]
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K. A. Whartenby, E. E. Straley, H. Kim, F. Racke, V. Tanavde, K. S. Gorski, L. Cheng, D. M. Pardoll, and C. I. Civin Transduction of donor hematopoietic stem-progenitor cells with Fas ligand enhanced short-term engraftment in a murine model of allogeneic bone marrow transplantation Blood, October 16, 2002; 100(9): 3147 - 3154. [Abstract] [Full Text] [PDF]
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C. A. Bonham, L. Peng, X. Liang, Z. Chen, L. Wang, L. Ma, H. Hackstein, P. D. Robbins, A. W. Thomson, J. J. Fung, et al. Marked Prolongation of Cardiac Allograft Survival by Dendritic Cells Genetically Engineered with NF-{kappa}B Oligodeoxyribonucleotide Decoys and Adenoviral Vectors Encoding CTLA4-Ig J. Immunol., September 15, 2002; 169(6): 3382 - 3391. [Abstract] [Full Text] [PDF]
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 M. F. Lipscomb and B. J. Masten Dendritic Cells: Immune Regulators in Health and Disease Physiol Rev, January 1, 2002; 82(1): 97 - 130. [Abstract] [Full Text] [PDF]
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H. Matsue, K. Matsue, M. Kusuhara, T. Kumamoto, K. Okumura, H. Yagita, and A. Takashima Immunosuppressive properties of CD95L-transduced "killer" hybrids created by fusing donor- and recipient-derived dendritic cells Blood, December 1, 2001; 98(12): 3465 - 3472. [Abstract] [Full Text] [PDF]
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S. H. Kim, S. Kim, C. H. Evans, S. C. Ghivizzani, T. Oligino, and P. D. Robbins Effective Treatment of Established Murine Collagen-Induced Arthritis by Systemic Administration of Dendritic Cells Genetically Modified to Express IL-4 J. Immunol., March 1, 2001; 166(5): 3499 - 3505. [Abstract] [Full Text] [PDF]
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R. M. Gorczynski, Z. Chen, D. A. Clark, J. Hu, G. Yu, X. Li, W. Tsang, and S. Hadidi Regulation of Gene Expression of Murine MD-1 Regulates Subsequent T Cell Activation and Cytokine Production J. Immunol., August 15, 2000; 165(4): 1925 - 1932. [Abstract] [Full Text] [PDF]
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