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Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 1047-1055
IMMUNOBIOLOGY
From the Kimmel Cancer Institute, Jefferson Medical College,
Philadelphia, Pennsylvania.
In graft-versus-leukemia (GVL) responses, the cellular subsets and
effector mechanisms responsible for cytotoxicity against leukemic cells
in vivo remain poorly characterized. A murine model of
syngeneic GVL that features CD4+ and CD8+
T-cell responses against the MMB3.19 myeloid leukemia cell line has
been previously described. MMB3.19 expresses high levels of functional
Fas and tumor necrosis factor (TNF) receptors that do not transduce
proapoptotic signals. Through the use of perforin- and Fas ligand
(FasL)-deficient mice, it was demonstrated that CD4+ T
cells mediate anti-MMB3.19 effects in vivo primarily through the use of
FasL and secondarily through perforin mechanisms. Conversely, CD8+ T cells induce GVL effects primarily through the use
of perforin and minimally through FasL mechanisms. Although the in vivo
observations of CD8+ T cells were reflective of their in
vitro cytotoxic T lymphocyte (CTL) activity, for CD4+ T
cells, in vitro responses were dominated by the perforin pathway. In
addition, the diminished capacity of T cells from perforin- and
FasL-deficient mice to lyse MMB3.19 target cells appeared directly
related to their deficient cytotoxic functions rather than to defects
in activation because these cells were fully capable of mounting
proliferative responses to the tumor cells. These findings demonstrate
that GVL responses of T-cell subsets can involve preferential use of
different cytotoxic mechanisms. In particular, these
findings identify a role for both FasL-employing CD4+
CTLs and the more novel perforin-utilizing CD4+ T-cell
subset in responses against a myeloid leukemia.
(Blood. 2000;96:1047-1055)
Graft-versus-leukemia (GVL) responses are antitumor
effects exerted by donor cells following hematopoietic stem cell (HSC) transplantation (HCT). GVL responses have been observed in patients with myeloid and lymphoid leukemias,1,2 and similar
antitumor effects have been found in other malignancies such as
multiple myeloma2 and breast cancer.3 However,
myeloid leukemias seem particularly susceptible to GVL effects. T cells
play a major role in GVL activity; this is based on findings that
T-cell depletion of HSC grafts results in higher rates of leukemic
relapse,4-6 the isolation of patient-derived T-cell clones
that can kill leukemia cells in vitro,7-10 and the
observations of successful reversal of leukemia relapse after delayed
infusion of T cells.11
The target antigens to which a GVL response are directed remain
controversial and may likely involve both shared host alloantigens, which can induce graft-versus-host disease (GVHD), and unique tumor-specific antigens. Evidence for the alloreactive responses comes
from the clinical observation that leukemic relapse is less frequent in
patients suffering from acute or chronic forms of GVHD.12,13 In regard to leukemia antigen-specific
responses, investigators have reported cohorts of allogeneic HCT
patients who have experienced GVL without apparent GVHD.13
In addition, autologous and syngeneic HSC grafts can be manipulated to
induce GVL with or without a concomitant autoimmune-like
GVHD.14-19
T cells can mediate GVL effects through several means, depending upon
their subset functional capabilities, and can involve either cytokine
induction of other effector cells (eg, via inflammatory molecules such
as interferon- We have previously established a mouse model of syngeneic and,
therefore, tumor antigen-specific GVL activity that features CD4+ and CD8+ T-cell responses against
MMB3.19, a myeloid leukemia of C57Bl/6 (B6) origin.25 In
the present study, we demonstrate that GVL activity against MMB3.19 is
not mediated by tumor necrosis factor- Mice
Cell lines
Media Phosphate-buffered saline (PBS) supplemented with 0.1% bovine serum albumin (BSA) (Sigma Chemical Co, St Louis, MO) was used for all in vitro manipulations of the donor bone marrow and lymphocytes. Immediately prior to injection, the cells were washed and resuspended in PBS alone. For in vitro assays, the cells were cultured in RPMI 1640 supplemented with 10% FBS, 5.5 × 10 5
mol/L 2-mercaptoethanol, 2 mmol/L L-glutamine, 50 IU/mL penicillin, and 50 µg/mL streptomycin.
Irradiation All recipient mice received an 850- or 950-cGy exposure from a Mark-I-68A cesium 137 (137Cs) source (JL Shepherd and Assocs, San Fernando, CA) at 143 cGy/min as indicated. For in vitro assays, MMB3.19 cells received a 30-Gy exposure.Monoclonal antibodies Ascitic fluids containing mAbs (clone names in parentheses; American Type Culture Collection, Manassas, VA) specific for Thy-1.2 (J1j), CD4 (RL172), CD8(3.168), natural killer 1.1 (NK1.1) (PK136), and rat immunoglobulin G (IgG) (MAR18.5; gift of Dr Robert Levy, Univ of Miami, Miami, FL) were used for the preparation of cellular grafts and culture supernatants. Other mAbs used included the supernatant of the rat-antimurine B220 14.8 mAb (gift of Dr Levy); affinity-purified goat antimouse IgG (whole molecule) antibodies (Cappel, Cosa Mesa, CA); guinea pig serum, which was prepared in our laboratory or purchased (Rockland, Boyertown, PA) and used as a source of complement for all mAb treatments; and biotinylated anti-Fas mAb (Jo2; PharMingen, San Diego, CA). For the phenotyping of MMB3.19 cells and the monitoring of cellular subset depletions from transplant grafts, we used fluorescein isothiocyanate (FITC)- and phycoerythrin (PE)-conjugated versions (clone names in parentheses, PharMingen) of the following mAbs that were specific for CD3 (145-2C11), CD4
(RM4-5), CD8 (53-6.7), B220 (RA3-6B2), 2B4, and an isotype control
(R35-95). We also used streptavidin (SA)-PE (Caltag, South San
Francisco, CA).
Flow cytometry Appropriate mAbs in volumes of 25 µL were incubated with 2-5 × 105 cells in the wells of a 96-well U-bottom microplate at 4°C for 25 minutes, centrifuged at 1500 rpm for 3 minutes, and washed with PBS containing 0.1% BSA and 0.01% sodium azide (wash buffer). When applicable, SA-PE or a secondary antibody was added in a volume of 25 µL at 4°C for 25 minutes, followed by 2 washes with wash buffer. The fluorescence analysis was performed on an EPICS Profile II analyzer (Coulter, Hialeah, FL) in the Kimmel Cancer Institute Flow Cytometry Facility, Philadelphia, PA.Preparation of cellular grafts Bone marrow cells were obtained from the femurs and tibias of wt mice by flushing with PBS. To prepare anti-Thy-1-treated (T-cell-depleted) bone marrow (ATBM), the cells were incubated with J1j mAb (at 1:100 dilution) and complement (at 1:25 dilution) at 37°C for 45 minutes and were washed extensively. T-cell-enriched donor cell populations were prepared by treating pooled spleen and lymph node cells in 2 steps: (1) Gey's balanced salt lysing solution containing 0.7% ammonium chloride (NH4Cl) was used to remove red blood cells (RBCs), and (2) panning on a plastic petri dish precoated with a 5 µg/mL dilution of goat antimouse IgG for 1 hour at 4°C was used to remove B cells. These treatments resulted in donor populations of 90%-95% CD3+ cells, as quantitated by fluorescent flow cytometry. As required, the suspensions of enriched T cells or unfractionated splenocytes were depleted of CD4+ or CD8+ cells by treatment with the appropriate mAbs and complement at 37°C for 45 minutes. At the same time, B220+ B cells and T cells were depleted by the addition of 14.8 culture supernatant and MAR18.5 ascites to the cellular preparations. These treatments reduced the targeted populations down to background levels on flow cytometric analysis.Cytotoxic assays Mice were presensitized with 5 × 106 irradiated (30 Gy) MMB3.19 cells intraperitoneally (i.p.), and splenocytes or lymph node cells were harvested after 2 weeks and restimulated in vitro for 5 days with irradiated MMB3.19 cells and 1:20 of T-STIM culture supplement (Becton Dickinson Labware, Franklin Lakes, NJ). CD4+ or CD8+ T cells were prepared by depleting splenocytes or lymph node cells of the other T-cell subset and NK cells. The JAM assay was used to measure cytotoxicity.27 Briefly, MMB3.19 cells growing in the log phase were subcultured at 5 × 105 cells per mL in media containing 0.0925 Bq/mL (2.5 µCi/mL) of 3H-thymidine (TdR) and allowed to label for 4 hours. The cells were then washed with media and cultured in 96-well U-bottom plates at 1 × 104 cells per well, along with effector cells at varying numbers. After 4 or 8 hours as indicated, the degree of target-cell DNA fragmentation was determined by comparing the radioactive incorporation of wells containing only target cells to wells containing both target and effector cells.Survival assay for GVL activity One day prior to transplantation, recipient mice were challenged with an i.p. injection of 0.5 mL PBS with or without 105 MMB3.19 cells. On the following day, recipient mice were lethally irradiated with 8.5 or 9.5 Gy, and approximately 6 hours later, they were injected intravenously (i.v.) with either 2 × 106 donor ATBM cells alone as a negative control or a mixture of ATBM cells plus manipulated donor lymphocyte populations. The mice were checked daily for morbidity and mortality until the experiments were terminated. As indicated, the data were pooled from 2 separate experiments, and median survival times (MSTs) were determined. Statistical comparisons between experimental groups were performed by the nonparametric Wilcoxon signed rank test.RT-PCR Total cellular RNA was prepared from 106-107 MMB3.19 cells by homogenization in 1 mL Ultraspec (Biotecx Laboratories, Houston, TX), separated of cellular DNA and protein by the addition of a 1:5 volume of chloroform, vortexed for 5 seconds, and centrifuged at 12 500 rpm for 15 minutes. The aqueous phase was transferred to a clean Eppendorf tube, and the RNA was precipitated at 4°C by the addition of an equal volume of isopropanol and then centrifuged at 12 500 rpm for 15 minutes. The pellet was washed with 75% ethanol in diethyl pyrocarbonate (DEPC)-treated water and centrifuged again. The RNA pellet was resuspended in 25 µL DEPC water, heated to 55-65°C for 10 minutes, and stored at 20°C. Recovery of RNA was determined by
spectrophotometry. For each sample, the reverse transcription and PCR
reactions were performed in a 1-tube format using Ready-To-Go RT-PCR
Beads (Amersham Pharmacia Biotech, Arlington Heights, IL). The
following were added to each reaction: 1 µmol/L sense and antisense
primers, 1.5 µg oligo d(T), and 2-4 µg total RNA.
Fas sensitivity assays MMB3.19 cells were cultured in 40 mL medium (RPMI 1640 supplemented with 10% FBS, 5.5 × 105 mol/L
2-mercaptoethanol, 2 mmol/L L-glutamine, 50 IU/mL penicillin, and 50 µg/mL streptomycin) in tissue culture flasks at
1 × 105 cells per mL with either 500 ng/mL anti-Fas mAb
(Jo2) or hamster anti-trinitrophenol (TNP) mAb as an isotype control.
Both antibodies (PharMingen) were obtained in a low-endotoxin, sodium
azide-free format. After 24 hours, the MMB3.19 cells were harvested,
and an aliquot was analyzed for viability by trypan blue staining. The
remainder of the cells were stained and analyzed using the APO-DIRECT
Kit (PharMingen), a flow cytometry-based TUNEL method (terminal
deoxynucleotidyl transferase-mediated dUTP nickend-labeling method).
Proliferation assays Pfpo, gld, and wt mice were injected with 5 × 106 irradiated (30 Gy) MMB3.19 cells, and their splenocytes were harvested 2 weeks later. The cells were cultured in triplicate at 2 × 105 cells per well in 96-well flat-bottom plates, and irradiated (30 Gy) MMB3.19 cells were added at 100 cells per well to half the samples. After 3, 4, and 5 days, the cells were harvested following an overnight pulse with 0.037 MBq (1 µCi) per well of TdR, and the radioactivity incorporation into DNA was measured. Stimulation indices were calculated by dividing the mean cpm of splenocytes cultured with MMB3.19 cells by the mean cpm of splenocytes cultured alone.TNF sensitivity assays Assay conditions to determine the sensitivity of MMB3.19 cells to exogenous TNF-mediated cytotoxicity were based on previous studies.28-29 Recombinant murine TNF- was used
(Peprotech, Rocky Hill, NJ). MMB3.19 and WEHI164 cells were cultured
overnight in quadruplicate in 96-well flat-bottom plates at
2 × 104 cells per well in a total volume of 100 µL.
We then added 100 µL media, containing either actinomycin D alone
(0.5 µg/mL final concentration) or actinomycin D and titrated
concentrations of TNF-, to each well. After 22 hours, 70 µL was
aspirated from each well prior to the addition of 50 µL
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (final concentration 1 mg/mL). After a 3.5- to 4-hour incubation at 37°C, 50 µL media was aspirated, and 150 µL
acidified isopropanol (0.04 N hydrochloric acid [HCl]) was added to
each well. The media in each well was then extensively run through a
pipette to dissolve crystals prior to the measurement of each well's
absorbance at 540 nm.
MMB3.19 cells express high levels of functional Fas Since FasL-Fas interactions are a major mechanism by which T cells mediate target cell death, it was first determined whether MMB3.19 cells had Fas molecules on their surface. Flow cytometric analysis revealed a high level of Fas expression, with 86.6% of the cells staining positive for the marker (relative mean fluorescence intensity of 14.85; Figure 1). Next, to determine whether the Fas molecules expressed by MMB3.19 cells were competent to receive death signals, the cells were incubated with anti-Fas mAb for 24 hours, and viability was measured by trypan blue exclusion. Anti-Fas mAb-treated tumor cells exhibited 22.2% nonviability compared to only 3.2% nonviability of control mAb-treated cells (Figure 2A). Furthermore, there were approximately 42% fewer viable cells remaining in the anti-Fas mAb-treated group (4.14 × 106 cells vs 7.13 × 106 cells). TUNEL analysis of the same cultures revealed that anti-Fas mAb treatment increased the percentage of apoptotic cells from 10.6% to 38.7% at the 24-hour time-point (Figure 2B). Collectively, this data indicated that MMB3.19 cells displayed sufficient levels of cell-surface Fas molecules that were capable of transducing apoptotic signals.
MMB3.19 expression of TNF receptors To determine whether MMB3.19 cells expressed TNF receptors, which could potentially be used as death-signaling molecules and are often expressed at low levels, RT-PCR analyses of both TNF receptor type I and II were performed (Figure 3). Synthesis of both TNF receptors was detectable in the MMB3.19 cells, and expression of each was comparable to that found for the TNF-sensitive cell line, WEHI164. However, despite the presence of the TNF receptors, the MMB3.19 cells were resistant to exogenous TNF-mediated cytotoxicity throughout a concentration range of 0.02-20 ng/mL. This is in contrast to WEHI164 cells, which were highly susceptible at all concentrations (Figure 4). In addition, RT-PCR analysis also revealed that MMB3.19 cells produced endogenous TNF- (data not
shown), which is consistent with the function of this cytokine as a
growth and/or activation factor. These combined results suggested that MMB3.19 cells do not receive death signals via ligation of their TNF
receptors with exogenous TNF-.
GVL-mediating T-cell subsets rely differentially on FasL and perforin The cytotoxic effector mechanisms by which T-cell subsets mediate syngeneic GVL responses to MMB3.19 leukemia challenge were investigated. Donor T cells from FasL- and perforin-deficient, mice along with 2 × 106 ATBM cells, were transplanted into lethally irradiated (8.5 Gy) B6 mice that were preinoculated with 1 × 105 leukemia cells. Mice infused with 2 × 106 CD4+ T cells from MMB3.19-presensitized B6 wt donors exhibited GVL activity with a significant (P .03) extension of MST to 43 days. This
contrasts with the 22-day MST for ATBM control mice that were
challenged with MMB3.19 cells but did not receive any donor T cells
(Figure 5A). Animals that were administered
2 × 106 CD4+ T cells from
MMB3.19-presensitized B6 pfpo donor mice
exhibited intermediate and marginally significant (P .075)
GVL activity, with an MST of 33 days. Furthermore, the survival pattern
of the pfpo group was significantly less
(P .02) than the wt control. In contrast,
2 × 106 CD4+ T cells from
MMB3.19-presensitized B6 gld donors did not appear to
mediate any GVL activity because recipients of these cells had an MST of 22 days, which is equivalent to the recipients of ATBM
plus leukemia cell challenge alone (P  .38). The levels of
GVL activity also correlated with the proportion of surviving mice at the termination of experiments on day 46; ie, 40% for mice
receiving wt CD4+ T cells, 20% for recipients of
pfpo cells, and 0% for mice
administered gld cells.
MMB3.19-directed CTL activity in vitro reflects GVL activity in
vivo
Splenocytes from wt, gld, and
pfpo mice can proliferate comparably in
response to MMB3.19 stimulation
GVL responses can potentially be mediated by T cells
present in both autologous and allogeneic HSC grafts. Interestingly, the myeloid leukemias seem most sensitive to GVL activity, although very little is known about these responses. Investigations into murine
GVL myeloid models have been hampered in the past by the limited
availability of myeloid cell lines, particularly those that do not
involve the expression of retroviral antigens. Our model for GVL
responses features a myeloid leukemia that was transformed through
transduction with a c-myc construct that does not result in any
detectable virus-related protein
expression.25 In addition, c-myc is a frequently
mutated gene in hematologic malignancies, and therefore in contrast to
other cell lines,30-32 the leukemia-specific antigen(s)
expressed by the MMB3.19 cells may reflect the consequences of
oncogenesis rather than retroviral infection.
We express our gratitude to Jeffrey Vakil and Kristin Naper for
technical assistance and to David Dicker for his expertise in flow
cytometric analyses.
Submitted January 10, 2000; accepted March 31, 2000.
Supported by research grants R01 HL55593, R01 CA60630, and
T32 CA09683 from the U.S. Public Health Service.
Reprints: Robert Korngold, Kimmel Cancer Institute, Jefferson
Medical College, 233 South 10th Street, Philadelphia, PA 19107; e-mail:
r_korngold{at}lac.jci.tju.edu.
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.
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Abstract
Introduction
[IFN-
