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IMMUNOBIOLOGY
From the Departments of Laboratory Medicine, Pathology,
and Dermatology and the Section of Immunobiology, Yale University
School of Medicine, New Haven, CT; and the Department of Medicine,
Section of Hematology and Oncology, University of Pennsylvania School
of Medicine, Philadelphia, PA.
Graft-versus-host disease (GVHD) is a major cause of morbidity and
mortality of allogeneic stem cell transplantation. Strategies to
control GVHD while maintaining graft versus leukemia (GVL) include
herpes simplex virus thymidine kinase (HSV-tk) gene transduction of donor T cells followed by treatment with ganciclovir (GCV). Alternatively, GVHD and GVL may be mediated by distinct processes. In
this regard, whether cytokine polarization occurs and to what degrees
various subsets of cytokine-producing T cells mediate GVHD or GVL has
been an active area of research using cytokine or cytokine antibody
infusion or genetically deficient mice. This study takes a different
approach that allows simultaneous investigation into both the
mechanisms underlying GVHD reactions and the efficacy of HSV-tk suicide
gene-based T-cell deletion. A source of donor T cells, splenocytes from
mice transgenic for HSV-tk controlled by elements of either the
interleukin-2 (IL-2) or IL-4 promoters (IL-2-tk and IL-4-tk,
respectively) was used, thus allowing investigation into the roles of
T1 and T2 cells in ongoing GVHD reactions. To assess treatment rather
than prevention of GVHD, GCV was started at peak disease. Remarkably,
treatment at this late time point rescued mice from the clinical
effects of GVHD caused by T cells expressing either transgene. Thus,
both T1 and T2 cells play an important role in clinical GVHD in a minor
histocompatibility antigen-mismatched setting. In addition,
because clinical disease was reversible even at its maximum, these
observations provide controlled evidence that this strategy of
treating ongoing GVHD could be effective clinically.
(Blood. 2001;98:3367-3375) Graft-versus-host disease (GVHD) remains a major
cause of morbidity and mortality from allogeneic stem cell
transplantation (alloSCT). It thereby limits the application of this
therapy for the cure of hematopoietic stem cell disorders. In the
context of cancer therapy, GVHD is often accompanied by graft versus
leukemia (GVL), which is responsible for much of the antineoplastic
effect.1,2 The combination of toxic and desirable effects
of donor T cells creates a therapeutic paradox. It would be ideal to
devise methods of separating GVHD from GVL or to control GVHD,
balancing the benefits of GVL with the toxicity of GVHD. Much effort
has gone into various approaches to accomplish these goals, with
partial success.3-7
Nonetheless, ultimate success will require a better understanding of
the pathophysiology of GVHD and the mechanisms of GVL. An area of
active GVHD research is whether cytokine polarization occurs and to
what degrees various subsets of cytokine-producing T cells contribute
to disease.8-16 In particular, it has been suggested that
acute GVHD (aGVHD) is the result of T1 polarization, whereas chronic
GVHD may be due to outgrowth of T2-polarized donor T
cells.8,17-20 Indeed, there is some evidence that Th2
cells can inhibit aGVHD,11 although studies on this issue
have been equivocal. Cytokine reverse transcriptase-polymerase chain
reaction results from mice undergoing either aGVHD or chronic GVHD in a parent into F1 (P Other investigators have proposed strategies to gain control of GVHD or
to separate GVHD from GVL,24-30 most notably suicide gene
transduction.31-34 In this strategy, donor T cells express a gene, such as herpes simplex virus thymidine kinase (HSV-tk), which
renders the T cell sensitive to killing by ganciclovir (GCV), provided
the cell is also dividing. To apply this strategy clinically, a method
of efficient gene transduction is required, along with the ability to
select transduced cells. Furthermore, a number of biological issues
need to be worked out, including T-cell and GCV dosing schedule,
specificity/toxicity of GCV treatment, evaluation of GCV effect in
various target tissues, effect on T-cell immunocompetence after
treatment, and the reversibility of established GVHD
lesions.35 Nonetheless, this strategy has been implemented
in humans as well as in animal models. Indeed, promising although
anecdotal reports have suggested efficacy in humans, arguing that
further effort should be put into this approach.7,33
Similarly, a few murine models using T cells from mice that are
transgenic (Tg) for HSV-tk have been developed, which have largely been
used to show that treatment with GCV at the time of transplantation in
fully allogeneic models can prevent disease, although in one report,
there was partial efficacy in a treatment model.34,36,37
We have taken a somewhat different approach that allows simultaneous
investigation into both the mechanisms underlying GVHD reactions and
the feasibility of HSV-tk-based T-cell deletion. Our model also takes
advantage of Tg-based expression of HSV-tk, thus obviating any issues
of gene transduction. We have used Tg mice in which the HSV-tk
transgene is controlled by either the IL-2 or IL-4 promoters (IL-2-tk
and IL-4-tk, respectively), thus allowing investigation into the roles
of T1 and T2 cells in ongoing GVHD reactions.38-40 These
transgenes have been well studied in vitro. Although both are expressed
very early after T-cell activation, under conditions in which T cells
are polarized toward Th1 cytokine expression, only the IL-2-tk
transgene is expressed, whereas the opposite is true under Th2
polarization conditions.38-40 The use of conditional
ablation differs from other approaches to investigating cytokine
polarization in that (1) T-cell polarization is initiated normally, (2)
potential developmental abnormalities associated with KO mice are
avoided, and (3) deletion is more precise than cytokine infusion or
inhibition via antibodies that can affect multiple tissues and does not
necessarily mimic the effects of cytokine polarization. In addition,
inhibition or KO of particular cytokines may not completely inhibit the
biologic effect of a polarized cell (eg, IL-4 inhibition may leave the
effects of IL-5 unchecked, although it may also tend to polarize cells
away from the T2 type).
We have crossed these transgenes onto the B10.BR background and have
used the minor histocompatibility antigen (miHA)-mismatched, MHC-matched B10.BR Mice
GVHD induction
Osmotic pump implantation Recipients were randomly divided into 2 groups at day 14 to receive pumps containing GCV (625 mg/mL; 3.75 mg/d) (Roche Laboratories, Nutley, NJ) or PBS. Animals were anesthetized by injection with a mixture of ketamine (10 mg/mL) and xylazine (2 mg/mL) at a dose of 10 µL/g intraperitoneally. The pumps were implanted on the back under sterile conditions.Pathology techniques and staining Tissues (skin, ear, tongue, liver, and intestine) were harvested and divided into 2 parts. For hematoxylin and eosin (H&E) staining the organs were fixed in 10% formalin and embedded in paraffin. For immunohistochemistry, tissues were fixed with diluted formalin (containing 0.7% formaldehyde) overnight at 4°C, dehydrated in 30% sucrose for 3 hours, frozen in Tissuetek (Sakura, Elkhart, IN), and stored at 80°C.
Frozen tissues were sectioned and mounted on 10% polylysine-coated slides. After blocking with 10% normal rat serum (Gemini Bioproducts, Calabasas, CA) and 3% bovine serum albumin (BSA), sections were incubated with biotinylated anti-CD8 or -CD4 monoclonal antibodies (Pharmingen, San Diego, CA) overnight at 4°C. After washing with 1% BSA in PBS, slides were stained with streptavidin-conjugated horseradish peroxidase followed by development with 3-amino-9-ethyl-carbazole (Sigma, St Louis, MO). The sections were counter stained with hematoxylin and mounted with Glycergel (Dako, Carpinteria, CA). Sections were coded and scored without knowledge of the treatment group by pathologists (M.E.R. for gut and liver; J.M.M. for ear, skin, and tongue). Skin was scored 0 to 3 for interface/apoptosis, infiltration, fibrosis, and extent of disease and the scores were totalled. For the intestine, an integrated GVHD severity score was assigned (0 to 3) by the pathologist on the basis of the following parameters: apoptosis, reactive epithelium, and inflammation. Flow cytometry To determine the origin of lymphocytes in the lymphoid organs, single cell suspensions were stained with Cy5-anti-Thy1.2 (clone 30H12, purified and conjugated in our laboratory) and biotin-anti-Thy1.1 (clone 19E12, prepared in our laboratory, and is specific for the recipient) followed by streptavidin-conjugated phycoerythrin (PE). Four-color staining was performed (CD44-FITC, CD4-PE, CD8-QR, and CD62L-SA-APC) as described.43 CD44 (clone IM7) and CD62L (clone Mel-14) antibodies were prepared in our laboratory, and the others were obtained from Pharmingen. Cells were analyzed on a FACSCalibur (Becton Dickinson, Woodstock, VT). Intracellular cytokine staining was performed as described44 after culturing cells for 5 hours with phorbol myristic acid and ionomycin, including monensin during the last 2 hours of culture. Prior to permeabilization, dead cells were identified with ethidium monoazide45 and later gated out in the FL3 channel. The following antibodies (Pharmingen) were used: rat immunoglobulin (Ig)G1-PE (clone R3-34), rat IgG2b-APC (clone A95-1), anti-IFN- -PE (clone XMG1.2), anti-IL-4-PE (clone 11B11),
anti-IL-2-APC (clone JES6-5H4).
Statistics Differences between survival curves were assessed by Kaplan-Meier analysis. The significance of differences in weights between groups at individual time points and between percentages of cells in flow-activated cell sorter (FACS) gates was assessed by using the 2-tailed Student t test. The Mann-Whitney test was used to determine significance of histology score differences between groups.
Experimental design Our experiments had 2 goals: first, to use cell ablation to test the hypothesis that acute GVHD is a T1-polarized disease and, second, to model the treatment of aGVHD by GCV-mediated deletion of thymidine kinase-expressing T cells. To address the first aim, we used both the IL-2-tk and IL-4-tk Tg mice. Although both transgenes are expressed very early on in T-cell activation, under Th1-polarizing conditions, IFN- expression by IL-4-tk CD4 cells is unaffected by GCV treatment
after only 2 days in culture in vitro, whereas IL-4 production from the
same cells is continually sensitive to GCV. Thus, the IL-4-tk gene is a
sensitive and specific tool for the ablation of Th2 cells but not Th1
cells.38-40
To address the second aim GCV treatment prevents death and reverses clinical GVHD as measured by weight gain Figure 1 shows the results of 4 separate experiments in which death was the end point. IL-2-tk mice were studied in experiments 1, 2, and 4 (total of 55 treated mice) and IL-4-tk in experiments 1 and 3 (total of 28 treated mice). All experiments included BM alone and Tg-negative groups. In each case, treatment of IL-tk mice with GCV provided substantial improvement of clinical GVHD as demonstrated by weight gain (left panels). In most experiments, the treated mice eventually gained weight to match that of BM-only controls, and in every case weight equaled or exceeded pretransplantation weights. PBS-treated mice typically had weights of 80% or less of pretransplantation levels. Differences between GCV- and PBS-treated recipients of IL-tk T cells were significant on multiple days in every experiment. In contrast, GCV-treated recipients of T cells from Tg-negative mice had persistent weight loss. In fact, in all experiments except experiment 4, the recipients of Tg-negative T cells that were treated with GCV had greater weight loss than those treated with PBS, the reverse of the situation with recipients of Tg-positive T cells. This finding could reflect some degree of toxicity of GCV in these sick recipients of Tg-negative T cells that was nevertheless counterbalanced by relief of GVHD in the recipients of Tg-positive T cells.
With the use of mortality as an end point, a similar situation was seen, although only in those experiments in which GVHD was severe enough to cause substantial mortality in the PBS-treated controls (ie, experiment 1 for IL-2-tk recipients and experiment 3 for IL-4-tk recipients). Consistent with the weight gain of GCV-treated recipients of IL-tk T cells, there were very few deaths in these groups in any of the experiments. Thus, as measured by weight loss and mortality, ablation of cells expressing IL-2 or IL-4 was sufficient to substantially ameliorate aGVHD even when it was severe at the onset of treatment (ie, when mice had already lost more than 30% of original body weight, Figure 1). The potential mechanisms for this protection will be discussed further below. GCV treatment partially ameliorates histologic disease To determine the effects on histologic disease, additional transplantation experiments were performed in which all surviving mice were killed at day 28. The effect of GCV on weight was similar to the prior observational experiments, thus further replicating and corroborating these experiments (data not shown). Skin, ear, tongue, small and large intestine, and liver were evaluated (Tables 1 and 2 and Figure 2). Statistically significant improvements in pathology score in GCV-treated recipients of IL-tk Tg T cells were observed in some but not all organs. Disease was significantly reduced in the tongue of IL-2-tk recipients. In particular, disease was ameliorated in the large intestine in both the IL-2-tk and IL-4-tk T-cell recipients. Trends toward histologic improvement, as indicated by the median scores in Tables 1 and 2, were seen in several other organs, but they did not reach significance. Because a limited number of samples were subjected to pathologic analysis, the statistical power of the study to detect differences was somewhat limited.
Figure 2 provides examples of the histology in IL-4-tk recipients. The mouse treated with PBS demonstrates multiple apoptotic cells in the epithelia as well as increased infiltrate in the lamina propria. In contrast, both BM-alone and the GCV-treated mice demonstrate relatively normal histologic pictures with few or no apoptotic cells and a sparse, normal content of lymphocytes in the lamina propria. Staining for CD4 and CD8 revealed the mixed nature of the infiltrate and increased numbers of cells in the PBS-treated mouse, consistent with the H&E staining. There are numerous intraepithelial CD4 and CD8 cells as well. In contrast, although there are CD4 and CD8 cells present in both the BM-alone and GCV-treated mice, they were fewer in number and were more rarely found in the epithelial layer, particularly for CD4 cells that are not normally detected in this location but are clearly present in the PBS-treated mouse. Overall, we conclude that GCV treatment substantially ameliorated but did not eliminate histologic GVHD. Differences among organs were noted, but it is difficult to draw conclusions about their meaning given the relatively small numbers of mice that were evaluated. In addition, the clinical significance of these residual lesions is uncertain, as there is no way to determine whether they are active or represent residua of previously active lesions. Because GCV only kills dividing cells that express thymidine kinase, the lesions may have been visible histologically but may not have contained dividing cells. Nonetheless, this result suggests that actively dividing cells are critical to the maintenance of clinical disease. GCV treatment restores naive, activated, and memory T-cell populations Mice undergoing aGVHD demonstrate increases in CD44+ T cells, particularly of CD62Llo cells that include memory phenotype T cells.47 Concurrently, the fraction of naive, CD44lo/CD62Lhi cells is reduced. We therefore asked if treatment with GCV of aGVHD induced by IL-tk Tg T cells would correct these alterations in T-cell activation phenotype subsets. Indeed, GCV treatment normalized these subsets for both CD4 and CD8 T cells in both the IL-2-tk and IL-4-tk mice. The distribution of T cells in these subsets was the same in BM-alone controls as in GCV-treated mice (Table 3), whereas in PBS-treated mice there was a lower frequency of naive T cells and most notably a higher proportion of memory-type CD44hi/CD62Llo cells. There was no effect of GCV on these subsets in Tg-negative mice (not shown). These data provide further objective evidence of the effect of GCV in reversing ongoing GVHD and are consistent with the effect occurring directly on T cells.
GCV treatment results in selective depletion of cytokine-secreting cells Because treatment of mice that received IL-tk Tg T cells led to amelioration of disease, one might expect that, at least in some organs, there would be a discernible effect of GCV treatment on the frequency of cytokine-secreting cells. To search for this effect, we killed mice 1 week after pump implantation (day 21 after transplantation), a time when the effect of GCV on weight is noticeable (Figure 1 and data not shown) and also when there are substantial cytokine-secreting cells (J.L. and M.J.S., unpublished observations and Figure 3, 2001) in untreated mice. Indeed, in recipients of IL-2-tk T cells, treatment for 1 week with GCV led to a significant decrease in IL-2-secreting cells in both spleen (P = .03) and liver (P = .008), as detected by FACS. The frequencies of IFN- were also
reduced in spleen (P = .019), but the frequency of
IL-4-secreting cells was not significantly altered. Interestingly, in
IL-4-tk T-cell recipients, the frequency of IL-4-secreting cells was
significantly reduced by GCV treatment, but only in liver (P = .03) and not in spleen (P = .13, Figure 3). Treatment of
IL-4-tk recipients with GCV did not affect IL-2- or IFN--secreting cell frequencies in either organ. These data provide evidence that GCV
does have a specific effect on the intended target cells and
indicate that the independent effects of IL-4-tk and IL-2-tk inhibition
do not work by cross-inhibition of the opposite cytokine. Because a
proportion of IL-2-secreting cells also express IFN-, it is not
unexpected that GCV treatment had a minor but significant effect on
these cells in spleen. The frequencies of donor T cells and of
cytokine-secreting donor T cells in mice receiving BM alone were
substantially lower than in T-cell recipients for all cytokines in all
tissues; their frequency was also unaffected by GCV treatment (data
not shown).
GCV treatment does not impair donor engraftment of T cells One potential drawback of GCV treatment in this setting is that it might lead to nonselective depletion of donor T cells. Unfortunately, congenic mice that would allow us to easily distinguish donor from recipient hematopoiesis are unavailable on these strains.48 Furthermore, in this model, engraftment of even T-cell-depleted BM is complete. Nevertheless, as B10.BR and AKR mice differ at the Thy1 allele, we were able to assess the development of donor-derived T cells via analysis of thymi and spleens (Table 4) of GCV-treated GVHD mice. Thymi of GCV-treated spleen cell recipients were much more cellular than PBS-treated mice at day 42 (data not shown), consistent with the clinical amelioration of GVHD in GCV-treated animals. The majority of thymocytes in GCV-treated GVHD mice was donor derived and had the typical thymic distribution of CD4 and CD8 staining (data not shown), suggesting that T-cell progenitor and likely pluripotent hematopoietic stem cell engraftment occurred in GCV-treated mice, as also found by others.49 The hypocellular thymi of PBS-treated mice precluded meaningful comparison with GCV-treated mice, but GCV if anything facilitated engraftment rather than hindered it in the thymus. Spleens at this point were predominantly populated with donor-derived T cells as well in all groups of mice (Table 4). When animals were killed at day 21, after 1 week of GCV and at a time when thymi were hypocellular in all mice, the majority of splenic T cells was Thy1.2+ and therefore donor in origin as well (data not shown). In these groups, there were substantially higher frequencies of donor-derived T cells in mice receiving BM plus T cells than BM alone, again indicating that mature T cells also engraft quantitatively in the presence or absence of GCV treatment. Thus, GCV treatment did not lead to the elimination of all mature donor T cells, again in accord with the idea that we selectively ablated cytokine-producing cells (Figure 3).
These studies had 2 main goals: to test the hypothesis that aGVHD is a T1-mediated disease and to determine the feasibility of GCV treatment of established severe aGVHD in a murine miHA-matched model. We addressed the first issue because cytokine polarization, which is known to control the phenotype of several autoimmune syndromes,50 could have important implications for the mechanism of GVH/L and its therapeutic manipulation. For example, polarization could explain the nature of lesions in various tissues and the differences between aGVHD and chronic GVHD.51 In addition, it has been proposed that control of polarization or selective inhibition of one T-cell cytokine subset may be a way to separate GVHD from GVL.3,51-54 We addressed the second goal, a model of suicide gene-based control of GVHD, in part because of its promising clinical utility.33,35 So far this has been demonstrated in only a limited way, and it is clear to us that animal models will be useful if not indispensable in truly understanding the mechanism of HSV-tk-based T-cell deletion and how it controls ongoing GVHD. To most closely mimic the clinical situation, we have used an MHC-matched, miHA-mismatched system and have administered GCV only after GVHD was well established. Clinical disease as measured by weight gain and survival regressed in GCV-treated IL-2-tk and IL-4-tk recipients. This clinical improvement correlated with partial amelioration of histologic disease, reduction in the frequency of the specifically targeted cytokine-secreting cells, and a decrease in memory T-cell accumulation, consistent with GCV-induced death of activated, dividing, alloreactive donor T cells. The significance of residual lesions and some residual cells expressing the "target" cytokine is difficult to assess. They could represent healing or inactive lesions. Alternatively, they could be the result of ongoing inflammation induced by (1) nondividing terminally differentiated T cells, (2) T cells no longer producing IL-4 or IL-2, or (3) processes that have become independent of the previously infused T cells. Finally, the lesions could be due to inefficiency in the GCV-mediated deletion of IL-tk T cells. Regarding the second goal, our data demonstrate that in a MHC-matched model, established GVHD can be reversed by using GCV ablation of tk-expressing T cells. It is important to emphasize that the evidence for this in humans thus far is anecdotal, limited to a few patients in an uncontrolled trial.7,33 Although these human trials are promising and in fact a rationale for our work, the extensive experiments shown here establish the efficacy of the strategy in a controlled way and, perhaps more importantly, provide a model for further investigation. There have been a few other reports on the use of tk-Tg mice and GCV for GVHD inhibition. In all but one report, GCV was given at the time of transplantation or shortly thereafter, thus providing a prevention and not a treatment model.34,37,46 In one case, GCV treatment was delayed.36 In that study, there was full MHC and miHA mismatch, a situation in which a very high frequency of both CD4 and CD8 cells is expected to be activated, unlike most human transplantations. It may be difficult to extrapolate this to the human miHA-mismatched or haplomismatched situation. Indeed, although some efficacy was shown in this full-mismatch model, mice remained symptomatic if GCV was delayed even until day 5 after transplantation, and, if delayed until 2 weeks, only partial protection was seen. Our studies are also informative on the roles of T1 and T2 cells in
aGVHD. This is a complex issue. It is well established that once a
T-cell clone is polarized, progeny of that clone maintain the T1 or T2
phenotype of the parent slt.39,40,55,56 For this reason,
we chose to treat our mice at day 14 when one would reasonably expect
that most mature donor T cells that were going to both survive 14 days
and see a recipient antigen strongly enough to contribute to GVHD would
have been activated. Indeed, infiltrating T cells In particular, the lack of effect of GCV treatment on IL-4-secreting cells in the IL-2-tk recipients argues against the possibility that manipulations designed to block either T1 or T2 cells are instead acting on T0 cells. Starting GCV treatment at day 14, when polarization has had enough time to occur, probably explains the specific effect. However, the issue of inhibiting T0 cells likely does apply in those strategies in which inhibition is constant from the beginning of allogeneic T-cell transfer, when a T0 phenotype would be expected among newly activated T cells.39,58,59 It is important to put our data in the context of other work on the
role of cytokine subsets in GVHD.38-40 The concept
that aGVHD is a T1-mediated syndrome (and chronic GVHD a T2-mediated syndrome) was initially proposed by Ferrara.9 They found
that Th2-polarized cells do not cause GVHD and, in fact, inhibit it when cotransferred with Th1 T cells10,11 and that IL-11
was found to promote T2 polarization and to reduce
GVHD.53,60 However, administration of IL-10, a cytokine
that is sometimes associated with T2 responses, either did not
prevent61 or else exacerbated62 aGVHD. IL-12,
a cytokine associated with T1 polarization, has had variable effects on
GVHD. During induction, neutralization of IL-12-inhibited GVHD in a
P More recently, using KO strategies, both Murphy et al22 and
Nikolic et al16 provided evidence that Th2 cells could be important. T cells from IFN- GCV/HSV-tk ablation differs from other strategies of cytokine
inhibition. In selective ablation, the T cells develop normally in the
donor, whereas this may not be the case in KO mice. In the IL-tk
system, after donor T-cell transfer to the recipient, disease evolves
normally, including initial T-cell polarization, until the point at
which GCV is given. This again might differ from the situation using KO
T cells. Ideally, the ablative approach deletes cytokine-producing T
cells and thus negates the effects of secreted cytokines as well as
other cytotoxic mechanisms such as FasL, granzyme, and tumor necrosing
factor One curious aspect of our results is that both types of T cells seem obligatory for clinical disease. In other GVHD models, either T1 or both subsets were shown to contribute, but there was still residual disease when one or the other was inhibited.16,22,23 This finding could reflect a difference in models. We use MHC-matched strains, in which donor T-cell activation is not likely as widespread as the MHC-mismatched models. Thus, the contribution from each T-cell cytokine subset may be relatively more important. The relatively complete inhibition of clinical disease with ablation of either IL-2- or IL-4-producing T cells could also reflect a difference in the mechanism of inhibition, with T-cell deletion perhaps being more efficient and thus showing a stronger phenotype. It is also possible that the 2 cell types are synergistic. There is precedent for this, including asthma,68-70 autoimmune gastritis,71 immune responses,72 tumor models,73 and in vitro.74,75 Clinical GVHD may be a threshold phenomenon, as is likely the case with frank clinical autoimmunity.76 This would be consistent with residual histopathology we observed in clinically well-appearing animals. That selective ablation of either T1 or T2 cells was enough to reverse
clinical GVHD in this system, raises the possibility that GVHD could be
treated while preserving some GVL. This is particularly tantalizing as
the cytokine secretion data suggest that ablation in the 2 different
settings are working at least in part via different mechanisms.
Polarization as an approach to maintain GVL while treating GVHD has
also been suggested by others.16,53,77 GVHD proceeds via
multiple mechanisms, at least involving FasL, perforin/granzyme,
TNF-
We thank Ann Haberman for critical reading of the manuscript and Richard Flavell for donating the tk transgenic mice.
Submitted July 31, 2000; accepted July 26, 2001.
Supported by grant P50HL54516 from the National Institutes of Health. J.L. is a Leukemia and Lymphoma Society Postdoctoral Fellow. W.D.S. is supported by NIH grant K08-HL03979.
W.D.S. and M.J.S. are cosenior authors.
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: Mark J. Shlomchik, Department of Laboratory Medicine, Rm CB465, Yale University School of Medicine, 333 Cedar St, Box 208035, New Haven, CT 06520-8035; e-mail: mark.shlomchik{at}yale.edu.
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Murphy WJ, Welniak LA, Taub DD, et al.
Differential effects of the absence of interferon- |
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Abstract
Introduction
F1) model have been
supportive.
modeling of GCV treatment, rather than
prevention,
(TNF-