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TRANSPLANTATION
From the University of Minnesota Cancer Center and
Department of Pediatrics, Division of Bone Marrow Transplantation,
Minneapolis; Kimmel Cancer Institute, Thomas Jefferson Medical College,
Philadelphia, PA; Department of Microbiology, Dartmouth Medical
College, Hanover, NH.
We previously reported that ex vivo blockade of the CD40:CD40L
costimulatory pathway in primary mixed lymphocyte reaction cultures resulted in profound in vitro secondary hyporesponsiveness and
30-fold or greater protection from graft-versus-host-disease (GVHD)
lethality. Present studies demonstrate that tolerance induction via
costimulatory blockade also results in the generation of a potent
immunoregulatory cell that inhibits both naive and primed alloresponses. The immunoregulatory capacity was dependent upon cell-to-cell contact that prevented the full activation of the naive or
primed cells. The inhibitory effect of tolerized cells did not preclude
the response of naive T cells to nominal protein antigen if antigen was
present at high concentration. However, under suboptimal antigen
concentration, nonspecific inhibition of responses occurred. The
tolerized regulatory cell population maintained a polyclonal T-cell
receptor V Bone-marrow transplantation is increasingly used
for the treatment of a number of malignant and nonmalignant disorders
of both hematologic and nonhematologic origin. Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality after
transplantation. Although GVHD can be prevented by rigorous ex vivo
T-cell depletion of the donor graft or prolonged global immunosuppression of the recipient, these strategies increase the rates
of leukemic relapse, infections, and graft failure. A tolerization
strategy that would selectively target only that small fraction of
potentially alloreactive T cells but preserve beneficial T cells would
be desirable. An ex vivo costimulatory blockade strategy is a
potentially attractive candidate for clinical application for the
prevention of GVHD.
We reported previously that ex vivo blockade of the CD40:CD40L
costimulatory pathway in murine CD4+ T cells in mixed
lymphocyte reaction (MLR) culture results in profound primary
and secondary hyporesponsiveness and 30-fold or greater protection from
GVHD lethality.1 These results fit well with the accepted
2-signal model of T-cell activation.2,3 Productive T-cell
activation and proliferation require 2 signaling events. Signal 1 is
the engagement of the T-cell receptor (TCR) with the
MHC-peptide ligand complex on the surface of the
antigen-presenting cell (APC). Additional costimulatory signals (signal
2) are required for the full activation of the intracellular signaling
cascade, interleukin-2 (IL-2) production, and T-cell proliferation.
Signal 1 in the absence of signal 2 renders a T-cell anergic or
hyporesponsive upon antigen restimulation.
The novel finding of these studies is that T cells tolerized to
alloantigenic stimulators via ex vivo CD40:CD40L blockade acquire
potent regulatory function capable of inhibiting both naive and
antigen-experienced T cells. While other investigators have reported
that anergic T cells can acquire regulatory capacity,4-6 the anergy-inducing strategies in those studies did not involve costimulatory blockade and this would not necessarily be expected to
result in the same phenomenon. Moreover, the acquisition of regulatory capacity as a result of ex vivo costimulatory blockade is
not readily predictable from the 2-signal model of T-cell activation. The regulatory function of the tolerized cells requires cell-to-cell contact that prevents the full activation of the naive or primed cells.
Tolerized cells do not require their obligate antigen for their
suppressor function. The regulatory capacity of tolerized cells induced
by CD40:CD40L costimulatory pathway blockade is not completely
specific, but vigorous T-cell responses to neoprotein antigens can
occur under optimal antigen conditions.
These studies implicate both tolerance induction and the generation of
regulatory function as separate factors contributing to the profound
degree of GVHD protection observed after the in vivo adoptive transfer
of tolerized cells into irradiated recipients. These data further
suggest that tolerized cells may be useful both for GVHD prevention and therapy.
Mice
In vitro MLR cultures
GVHD induction The bm12 recipients were sublethally irradiated with 6 Gy total body irradiation from a 137Cesium source 4 hours prior to cell infusion. Then, 105 freshly purified naive CD4+ cells or day-10 cultured, primed control cells were injected intravenously. Cohorts of mice received a second intravenous injection of 105 anti-CD40L-tolerized cells immediately following the injection of naive or primed cells. Peripheral blood was obtained by retro-orbital venipuncture on day 14 for measurement of hematocrit (HCT) values as an indicator of the bone marrow-destructive effects of infused T cells. HCTs were also determined at the end of the observation period of 2 months. Owing to the sublethal irradiation dose and relatively low numbers of donor T cells infused, surviving mice recover with host-type T cells, and infused donor T cells do not persist long term. Mice were monitored daily for survival and weighed twice weekly as well as examined for the clinical appearance of GVHD.Flow cytometry Cells were assessed for evidence of blastogenesis and activation by forward scatter (FSC) and side scatter (SSC) profiles and the coexpression of CD4 and activation antigens, including CD25, and L selectin (CD62L). Congenic B6 naive and tolerized cells from coculture were distinguished by CD45.1, and CD45.2. KJ1-26 (anti-clonotypic mAb) was used to distinguish DO11.10.10 CD4+ cells. All studies were performed with 3-color flow cytometry with the use of fluorescein- and phycoerythrin-conjugated mAb (PharMingen). Fewer than 5% of cells in the live gate were identified as apoptotic by 7-aminoactinomycin (7AAD) (Sigma). Results were obtained by means of CellQuest software on a FACSCalibur (Becton Dickinson, San Jose, CA). FSC and SSC settings were gated to exclude debris and dead cells. A total of 104 cells were analyzed for each determination.V  b), purchased from Applied
Biosystems (Foster City, CA), and 17 different V family-specific
primers (V 1 through V16 and V18). The fluorescently labeled
PCR products were run on a sequencing gel and analyzed by Genotyper
Genescan software program (PE-Applied Biosystems).
Quantitation of cytokine levels by enzyme-linked immunosorbent assay Murine TH1 (IL-2 and interferon- [IFN-) and
TH2 (IL-4, IL-10, and IL-13) cytokine levels in the
supernatant of MLR cultures were quantitated by enzyme-linked
immunosorbent assay (R&D Systems, Minneapolis, MN). Sensitivity of the
assays was 4 pg/mL or less for each assay, except for IL-13 which was
22 pg/mL. A standard curve using recombinant protein was generated with
each assay.
Statistics Survival data were analyzed by life-table methods, and actuarial survival rates are shown. Group comparisons were made by log-rank test statistics. For other data, group comparisons were made by Student t test. P .05 was considered significant.
Anti-CD40L mAb-tolerized CD4+ T cells down-regulate a naive alloresponse both in vitro and in vivo A 10-day blockade of the CD40:CD40L costimulatory pathway in murine CD4+ T cells results in profound in vitro hyporesponsiveness and GVHD prevention.1 We reported that cells tolerized via ex vivo blockade of the CD40:CD40L pathway had evidence of activation as determined by flow cytometry and analysis of intracellular biochemical pathways.8 These data suggested that tolerized cells might have gained, rather than strictly lost, function. Additionally, other investigators have reported the acquisition of regulatory function in tolerized cells. Therefore, we evaluated the anti-CD40L mAb-tolerized cultures for regulatory potential. To determine the effect of tolerized cells on a naive alloresponse, B6 CD4+ cells, previously tolerized to bm12 alloantigen by exposure of a 10-day MLR culture to anti-CD40L mAb, were washed to remove Ab and cytokines and were added to a naive B6 CD4+ MLR culture containing bm12 splenic stimulators. Figure 1 illustrates the proliferative responses in the culture. By day 3 of culture, the addition of 1 tolerized cell to 3 naive CD4+ T cells reduced the naive alloresponse by over 80%. At the time of peak response in the control culture on day 4, the addition of tolerized cells inhibited the response by 94%. As few as 1 tolerized cell to 10 naive CD4+ T cells down-regulated the naive alloresponse equally potently (data not shown).
To determine whether this profound in vitro regulatory effect would
translate into protection from GVHD lethality in vivo, a uniformly
lethal dose of naive B6 CD4+ cells was infused into
sublethally irradiated bm12 recipients. The infusion of 105
naive B6 CD4+ T cells resulted in GVHD-induced bone marrow
aplasia, killing all control mice by 3 weeks after infusion of cells
(Figure 2A). In contrast, 75% of mice
receiving 105 naive CD4+ T cells and
105 tolerized cells administered by separate injections
survived the 2-month observation period (P < .001). As an
indicator of donor CD4+ T-cell-mediated GVHD-induced bone
marrow aplasia, HCT values were assessed in all mice on day 14 after
transfer of cells. In one representative experiment, the HCT was
16.2% ± 5.6% in mice receiving naive CD4+ cells versus
32.1% ± 2.4% in mice receiving both naive and tolerized CD4+ cells (P < .001). At the time that mice
were electively killed, at 2 months after transfer of cells, all
survivors of naive and tolerized cells had recovered HCTs to normal
values of 40% or higher. Clinically, survivors appeared GVHD-free and
had weights exceeding their pretransplantation weight (data not shown).
The in vitro effect of tolerized cells on a primed alloresponse was difficult to evaluate since primed cells exhibited a vigorous and rapid proliferative response upon alloantigen restimulation (data not shown). Therefore, an in vivo model was used to evaluate whether tolerized cells could down-regulate a primed alloresponse. To determine the effect of tolerized cells on a primed response in vivo, 105 primed cells with or without a second injection containing 105 tolerized cells were injected into sublethally irradiated bm12 recipients. All mice receiving only primed cells died of bone marrow aplasia by day 20 (Figure 2B). In contrast, mice receiving both primed and tolerized cells had a delayed onset of GVHD mortality with approximately 20% long-term survivors (P < .001). In one representative experiment, mice receiving only primed cells had an average day-14 HCT of 13.5% ± 6.0% versus 33.5% ± 5.0% in recipients of both primed and tolerized cells (P < .001). The mice surviving the 2-month observation period normalized their HCT at the time they were electively killed. Collectively, these data indicate that cells tolerized by a 10-day ex vivo culture with anti-CD40L mAb have potent regulatory capabilities to inhibit both a naive and a primed alloresponse. Conditions required for the down-regulatory effect of tolerized cells on naive cells To determine if a soluble factor was implicated in the regulatory activity or if cell-to-cell contact was a requirement for the down-regulation induced by anti-CD40L mAb-tolerized cells, transwell culture inserts were used. Washed tolerized cells plated with fresh stimulators in the transwell chambers allowed for the diffusion of potentially suppressive soluble factors across the membrane into the culture containing the naive CD4+ T cells and allostimulators but denied direct cell-to-cell contact between the naive and the tolerized T cells. Transwell experiments determined that cell-to-cell contact was essential for inhibition of a naive alloresponse (Figure 3). The diffusion of putative soluble factors from the tolerized cells in the transwell had no significant effect on the proliferation of the naive cells in response to alloantigen. This is consistent with our previously published data in which the potentially immunosuppressive cytokines, IL-4, IL-10, transforming growth factor- (TGF-), and IL-13 were undetectable in the supernatants of the tolerized cultures examined throughout the 10-day primary tolerization procedure.9
Supernatants from the transwell cultures were evaluated for IL-2
levels. The production of IL-2, known to be an indicator of tolerance
induction, in the B6 anti-bm12 MLR was completely abrogated by day 4 of
culture by the addition of tolerized cells if cell-to-cell contact was present (Table 1). Consistent with the
proliferation data, if cell contact was denied, the diffusion of
soluble factors from the tolerized culture had no effect on the levels
of IL-2 detected in the supernatant.
To investigate the effects of tolerized T cells on the activation of
naive T cells, a primary MLR of B6 CD45.1 CD4+ T cells and
bm12 splenic stimulators was established with and without the addition
of 10-day ex vivo tolerized B6 CD45.2 CD4+ T cells. On day
5, the cells were phenotyped, and recovery and the degree of activation
were evaluated. Approximately the same percentage of naive
CD45.1+ T cells was recovered whether they had been
cocultured with tolerized cells or not (29% versus 35%).
Although recovery of naive cells was similar for the 2 groups,
the naive cells cocultured with tolerized cells did not exhibit the
same degree of blastogenesis and activation as those in the control
culture (Figure 4). The FSC and SSC of
the naive cells cocultured with tolerized cells indicated reduced
blastogenesis as compared with the control group. CD25 expression was
also reduced in the naive cells cocultured with tolerized cells.
Additionally, although L selectin (CD62L) had significantly
down-regulated in the naive cells in the control group, expression
remained high in those naive cells in the coculture, suggesting they
had a different activation profile, as we have previously reported in
tolerized versus naive cells. Perhaps surprisingly, 137% of the input
number of the tolerized cells in the coculture group was recovered. As
well as having high FSC and SSC, 90% of the recovered tolerized cells
were CD25+ (Figure 4). Although the tolerized cells had
several parameters of highly activated cells, they remained high for L
selectin. We conclude that tolerized cells inhibit the proliferation of naive cells in response to alloantigen by a mechanism that requires cell-to-cell contact that prevents the full activation of the naive
cells and inhibits their IL-2 production. It is unknown whether this
inhibition of a naive alloresponse is due to suppression or killing of
the small number of potentially alloreactive naive T cells in the bulk
population.
Although tolerized cells can exert their down-regulatory effect on naive T cells in an antigen-nonspecific manner, this inhibition can be overcome if high-affinity T cells receive sufficient TCR signals To determine the specificity of the down-regulatory effect of the tolerized cells, a different model was used that would allow evaluation of the effects of tolerized cells on naive transgenic T-cell responses to their obligate peptide antigen. BALB/c CD4+ cells were tolerized to B6 alloantigen via an 8-day culture with anti-CD40L mAb. Tolerized cells were washed to remove antibody and cytokines and added to a primary MLR of naive BALB/c CD4+ T cells and T-cell-depleted, irradiated B6 splenic stimulators. As expected, tolerized cells inhibited the naive alloresponse by day 3 of culture (Figure 5A). A ratio of 1 tolerized cell to 3 to 10 naive cells proved optimal for the down-regulation. Impressively, as few as 1 tolerized cell to 100 naive cells resulted in an approximately 50% inhibition of the peak naive alloresponse. To determine the specificity of the regulatory effect, BALB/c CD4+ cells tolerized to B6 alloantigen were added to naive DO11.10 CD4+ cells (1:1 ratio) and mixed with irradiated BALB/c APCs. OVA peptide was added at an optimal concentration of 5.0 µg/mL. Under these conditions, the alloantigen-tolerized BALB/c CD4+ cells did not inhibit the naive DO11.10 OVA response (Figure 5B). As further evidence of the specificity of the inhibitory effects of alloantigen-tolerized cells, the levels of both TH1 and TH2 cytokines in the supernatants of the culture were analyzed on day 3 and day 5 of culture. Tolerized cells profoundly inhibited the production of IL-2 and IFN- by naive BALB/c CD4+ cells in response to
alloantigen (Table 2). Low to
undetectable levels of TH2 cytokines were detected in the
allo-MLR. In contrast, the addition of tolerized cells had little
effect on either TH1 or TH2 cytokine production
by naive DO11.10 CD4+ cells in response to an optimal OVA
peptide concentration.
To rule out the possibility that the OVA peptide response was too vigorous to uncover a nonspecific regulatory effect by the tolerized cells, the model was modified to use CB6 F1 APCs. The use of CB6 F1 APCs to present OVA peptide resulted in a more delayed and lower peak proliferative response as compared with BALB/c APCs. The peak response with F1 APCs was on day 6 with approximately 20 000 cpm as compared with BALB/c APCs, which resulted in a day-4 or day-5 peak response of 80 000 cpm (Figure 5B-C). Importantly, the F1 APCs also provided relevant alloantigen to the tolerized cells in the event that this was a requirement for their regulatory capacity. These conditions should maximize the likelihood that a nonspecific down-regulation would be detected. Under these conditions and with the use of F1 APCs, the tolerized cells did not inhibit a naive DO11.10 OVA response if OVA was present at a high concentration of 5.0 µg/mL (Figure 5C). It is important to note that DO11.10 mice have a high precursor frequency of high-affinity T cells for their obligate antigen, which generates responses that are probably difficult to suppress under high peptide concentration. However, if the concentration of OVA was reduced 10-fold to a suboptimal concentration of 0.5 µg/mL, the tolerized cells nonspecifically inhibited the naive DO11.10 response by 62% on day 5 and 87% on day 6 of culture (Figure 5D). In a different nontransgenic model, nonspecific inhibition of a naive
alloresponse was also noted. B6 CD4+ cells tolerized to
bm12 alloantigen potently inhibited a naive B6 CD4+
response to B10.BR (H2k) stimulators (Figure
6). It is important to note that unlike the DO11.10 transgenic model, a B6 anti-B10.BR response is a polyclonal T-cell response composed of low-, intermediate-, and high-affinity alloresponsive T cells of a relatively low precursor frequency and,
therefore, might be expected to be more easily suppressed. Together,
these data suggest that if the net TCR signal of the bulk T-cell
population is sufficiently low, owing to either a low frequency of
antigen-reactive T cells or a low concentration of neoprotein
antigen, then tolerized cells may nonspecifically inhibit a naive or
primed T-cell response. In the case of high concentrations of
neoprotein antigen and a high frequency of antigen-reactive T cells,
nonspecific down-regulation may be less likely to occur.
Tolerized cells retain a relatively broad TCR V repertoire, PCR-based TCR V CDR3-size spectratyping was performed on
10-day cultured primed and tolerized cells and compared with that of freshly purified CD4+ cells. V spectratype analysis of
17 different V families indicated that anti-CD40L-tolerized cells
retained a relatively broad V repertoire similar to control
CD4+ cells (Figure 7). In
contrast, primed cells exhibited V skewing and increased
oligoclonality within V families. These data indicate that
tolerization prevents the restricted V repertoire observed in
control primed cultures, potentially providing a broader array of T
cells that can respond to foreign pathogens and injured cells in
vivo.
CD4+CD25+ cells are required for the induction of tolerance and generation of regulatory capacity We have previously published that immune regulatory CD4+CD25+ T cells are required for tolerance induction via ex vivo costimulatory blockade.10 Depletion of CD4+CD25+ T cells from the CD4+ responder population completely abrogated ex vivo tolerance induction to alloantigen as measured by intact responses to alloantigen restimulation in vitro and lethal GVHD generation in vivo. To determine whether CD25+ cells were also required for the acquisition of regulatory capacity, whole B6 CD4+ T cells or B6 CD4+CD25 T cells and bm12 splenic stimulators
were cultured in the presence of anti-CD40L mAb. At the end of the
9-day MLR culture, cells were washed and coinjected with a lethal dose
of naive CD4+ T cells (Figure
8). Recipients of 105 naive
CD4+ T cells died by day 23 after infusion of
cells. Of the mice receiving 105 naive CD4+ T
cells and 105 anti-CD40L-cultured whole CD4+ T
cells, 63% survived the 2-month observation period. In contrast, recipients of naive CD4+ T cells and anti-CD40L-cultured
CD4+CD25 T cells died by 20 days after
infusion of cells. Thus, CD4+CD25+ immune
regulatory cells are required for the acquisition of the regulatory
capacity of anti-CD40L mAb-treated CD4+ T cells in MLR
cultures.
The novel finding of this study is that tolerance induction via ex
vivo blockade of the CD40:CD40L costimulatory pathway in allo-MLR
cultures results in the acquisition of potent suppressor function that
inhibits naive and primed alloresponses both in vitro and in vivo. This
immunoregulatory capacity requires cell-to-cell contact that prevents
the full activation of the naive (or primed) cells and inhibits their
production of IL-2 and IFN- The immunoregulatory nature of anergic T cells has been reported by several investigators. Taams et al6 reported that autoreactive rat CD4+ T-cell clones for experimental autoimmune encephalomyelitis and adjuvant arthritis could be induced to become anergic via T-cell presentation of the antigen. Anergic encephalitogenic cells specifically inhibited the in vitro peptide response of nonanergic cells of the same clone although they did not inhibit peptide responses of a different arthritogenic T-cell clone.6 However, anergic T-cell clones were able to nonspecifically suppress proliferative third-party responses of polyclonal lymph-node T cells provided that peptide recognized by the anergic T cells was present.6 Another group rendered human T-cell clones, specific for influenza hemagglutinin peptide, anergic either by incubation with antigen in the absence of APCs or by incubation with immobilized anti-CD3 mAb.4 Anergic T cells added to cultures containing potentially reactive T cells, APCs, and antigen inhibited proliferation in a titratable fashion. Their model also exhibited some, but not complete, specificity. Chai et al5 found that an alloreactive T-cell clone anergized in vitro via immobilized anti-CD3 mAb and then transferred in vivo into recipients of allogeneic skin grafts led to prolonged skin survival. Other investigators used in vivo tolerance-inducing strategies.11,12 Qin et al12 induced in vivo transplantation tolerance by transplanting skin from minor histocompatibility antigen-mismatched donors to recipient mice under the cover of nonlytic anti-CD4 and anti-CD8 mAbs. They demonstrated that immunoregulatory tolerant T cells in the host not only inhibited injected naive T cells from rejecting a new graft but also tolerized the injected naive cells. The studies presented in our paper extend the field by demonstrating the generation of a potent regulatory cell population by ex vivo costimulatory blockade of a single pathway in primary murine CD4+ T cells. It is interesting to note that the acquisition of regulatory capacity can occur in such varied tolerance-inducing strategies. It is tempting to speculate that tolerance and regulation may be inexorably linked. Despite the well-documented role of IL-4, IL-10, TGF- Analysis of CD45 congeneic naive and tolerized cells cocultured with
allostimulators for 5 days revealed similar recovery of the naive cells
regardless of whether tolerized cells were added to the culture despite
an almost complete inhibition of proliferation as measured by tritiated
thymidine uptake. The naive cells in the coculture phenotypically
appear to be less activated compared with those cultures in which the
tolerized cells have not been added. Additionally, the production of
IL-2 and IFN- Previous data indicate that tolerized cells are not only hyporesponsive in terms of proliferation and cytokine production upon alloantigen restimulation but also have greatly reduced cytolytic effector function, at least for their relevant target alloantigen.9 These data do not rule out the possibility that tolerized cells are mediating their regulatory function by killing the relatively small number of alloreactive T cells (a likely source of TH1 cytokines) and leaving the other T cells intact. Interestingly, tolerized cells added to the naive cells had undergone at least some proliferation since more tolerized cells were recovered than were originally added to the culture. Additionally, the tolerized cells had evidence of increased activation status, as suggested by a significant increase in FSC and SSC and the up-regulation of CD25 expression from approximately 50% to 90% in the course of the 5-day coculture (Figure 4 and data not shown). In contrast, tolerized cells showed no signs of activation upon alloantigen restimulation in secondary MLR in the absence of naive cells (data not shown). Thus, the activation of tolerized cells was increased by the presence of naive cells. CD4+ T cells tolerized to alloantigen inhibited the
response of a naive T cell to a neoprotein antigen under suboptimal,
but not optimal, TCR signaling conditions regardless of whether the relevant stimulators were present in the culture or not (Figure 6 and
data not shown). This may have important clinical implications if one
assumes the same is true in vivo. Although the nonspecific down-regulation of third-party T-cell responses associated with tolerization may seem to be clinically undesirable, we hypothesize that
some degree of nonspecificity will not preclude the in vivo generation
of beneficial T-cell responses in settings where TCR signaling is high
and the organism needs to respond. Consistent with this hypothesis,
various heterogeneous populations of suppressor or regulatory cells
occur normally in vivo and are essential to T-cell homeostasis and
maintenance of peripheral tolerance but do not abrogate the ability of
an animal to mount vigorous T-cell responses to antigen. The
maintenance of a relatively broad V We have previously published that CD4+CD25+
immune regulatory cells are required for tolerance induction via ex
vivo costimulatory blockade.10 These studies extend those
findings by demonstrating that tolerance induction results in
the generation of regulatory function. The mechanism by which this
CD4+CD25+ T-cell subset would positively
influence the induction of tolerance and the generation of regulatory
function is unknown. It is possible that the extended ex vivo
incubation with anti-CD40L mAb might preferentially enrich and
activate the CD4+CD25+ immune
regulatory population while deleting or blocking the activation and
proliferation of alloreactive CD25 In summary, we have shown that costimulatory blockade of an allo-MLR culture does not simply induce hyporesponsiveness to alloantigen restimulation but also actively induces cells with a potent regulatory capacity. We hypothesize that the acquisition of this suppressor function during tolerance induction may be harnessed to contribute to GVHD protection observed after the in vivo adoptive transfer of tolerized cells into irradiated transplant recipients. In the event that some transferred cells escape their tolerant state and become alloresponsive, other regulatory cells may be able to inhibit their clonal expansion and capacity to mediate GVHD. The infusion of tolerized cells as a means of preventing and treating GVHD warrants further investigation.
We would like to thank Dr Angela Panoskaltsis-Mortari and the University of Minnesota Cytokine Reference Laboratory for providing the cytokine data.
Submitted November 13, 2001; accepted February 4, 2002.
Supported by National Institutes of Health grants R01 AI 34495, 2R37 HL56067, R01 HL63452, and P01 AI-35225.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Bruce Blazar, University of Minnesota, MMC 109, 420 Delaware St SE, Minneapolis, MN 55455; e-mail: blaza001{at}tc.umn.edu.
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© 2002 by The American Society of Hematology.
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  M. Suzuki, X. Zheng, X. Zhang, M. Li, C. Vladau, T. E. Ichim, H. Sun, L. R. Min, B. Garcia, and W.-P. Min Novel Vaccination for Allergy through Gene Silencing of CD40 Using Small Interfering RNA J. Immunol., June 15, 2008; 180(12): 8461 - 8469. [Abstract] [Full Text] [PDF]
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 S. Mielke, R. Nunes, K. Rezvani, V. S. Fellowes, A. Venne, S. R. Solomon, Y. Fan, E. Gostick, D. A. Price, C. Scotto, et al. A clinical-scale selective allodepletion approach for the treatment of HLA-mismatched and matched donor-recipient pairs using expanded T lymphocytes as antigen-presenting cells and a TH9402-based photodepletion technique Blood, April 15, 2008; 111(8): 4392 - 4402. [Abstract] [Full Text] [PDF]
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S. J. Jenkins, G. Perona-Wright, A. G. F. Worsley, N. Ishii, and A. S. MacDonald Dendritic Cell Expression of OX40 Ligand Acts as a Costimulatory, Not Polarizing, Signal for Optimal Th2 Priming and Memory Induction In Vivo J. Immunol., September 15, 2007; 179(6): 3515 - 3523. [Abstract] [Full Text] [PDF]
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S. Mielke, K. Rezvani, B. N. Savani, R. Nunes, A. S. M. Yong, J. Schindler, R. Kurlander, V. Ghetie, E. J. Read, S. R. Solomon, et al. Reconstitution of FOXP3+ regulatory T cells (Tregs) after CD25-depleted allotransplantion in elderly patients and association with acute graft-versus-host disease Blood, September 1, 2007; 110(5): 1689 - 1697. [Abstract] [Full Text] [PDF]
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 B. R Blazar and W. J Murphy Bone marrow transplantation and approaches to avoid graft-versus-host disease (GVHD) Phil Trans R Soc B, September 29, 2005; 360(1461): 1747 - 1767. [Abstract] [Full Text] [PDF]
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L. M. Bagenstose, R. K. Agarwal, P. B. Silver, D. M. Harlan, S. C. Hoffmann, R. L. Kampen, C.-C. Chan, and R. R. Caspi Disruption of CD40/CD40-Ligand Interactions in a Retinal Autoimmunity Model Results in Protection without Tolerance J. Immunol., July 1, 2005; 175(1): 124 - 130. [Abstract] [Full Text] [PDF]
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B. Valzasina, C. Guiducci, H. Dislich, N. Killeen, A. D. Weinberg, and M. P. Colombo Triggering of OX40 (CD134) on CD4+CD25+ T cells blocks their inhibitory activity: a novel regulatory role for OX40 and its comparison with GITR Blood, April 1, 2005; 105(7): 2845 - 2851. [Abstract] [Full Text] [PDF]
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K. P. A. MacDonald, V. Rowe, A. D. Clouston, J. K. Welply, R. D. Kuns, J. L. M. Ferrara, R. Thomas, and G. R. Hill Cytokine Expanded Myeloid Precursors Function as Regulatory Antigen-Presenting Cells and Promote Tolerance through IL-10-Producing Regulatory T Cells J. Immunol., February 15, 2005; 174(4): 1841 - 1850. [Abstract] [Full Text] [PDF]
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L. Weiss, V. Donkova-Petrini, L. Caccavelli, M. Balbo, C. Carbonneil, and Y. Levy Human immunodeficiency virus-driven expansion of CD4+CD25+ regulatory T cells, which suppress HIV-specific CD4 T-cell responses in HIV-infected patients Blood, November 15, 2004; 104(10): 3249 - 3256. [Abstract] [Full Text] [PDF]
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L. Nagelkerken, I. Haspels, W. van Rijs, B. Blauw, J. L. Ferrant, D. M. Hess, E. A. Garber, F. R. Taylor, and L. C. Burkly FcR Interactions Do Not Play a Major Role in Inhibition of Experimental Autoimmune Encephalomyelitis by Anti-CD154 Monoclonal Antibodies J. Immunol., July 15, 2004; 173(2): 993 - 999. [Abstract] [Full Text] [PDF]
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S. Vigouroux, E. Yvon, E. Biagi, and M. K. Brenner Antigen-induced regulatory T cells Blood, July 1, 2004; 104(1): 26 - 33. [Abstract] [Full Text] [PDF]
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J. Vermeiren, J. L. Ceuppens, M. Van Ghelue, P. Witters, D. Bullens, H. W. Mages, R. A. Kroczek, and S. W. Van Gool Human T Cell Activation by Costimulatory Signal-Deficient Allogeneic Cells Induces Inducible Costimulator-Expressing Anergic T Cells with Regulatory Cell Activity J. Immunol., May 1, 2004; 172(9): 5371 - 5378. [Abstract] [Full Text] [PDF]
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Z.-m. Chen, M. J. O'Shaughnessy, I. Gramaglia, A. Panoskaltsis-Mortari, W. J. Murphy, S. Narula, M. G. Roncarolo, and B. R. Blazar IL-10 and TGF-{beta} induce alloreactive CD4+CD25- T cells to acquire regulatory cell function Blood, June 15, 2003; 101(12): 5076 - 5083. [Abstract] [Full Text] [PDF]
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C. Sharp, C. Thompson, E. T. Samy, R. Noelle, and K. S. K. Tung CD40 Ligand in Pathogenesis of Autoimmune Ovarian Disease of Day 3-Thymectomized Mice: Implication for CD40 Ligand Antibody Therapy J. Immunol., February 15, 2003; 170(4): 1667 - 1674. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
