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*
Department of Immunology and Division of Oncology, University of Washington, Seattle, WA 98195; and
Fred Hutchinson Cancer Research Center, Seattle, WA 98109
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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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Tolerance to self-proteins has been evaluated in several distinct murine models. Intrathymic expression of a self-protein usually leads to deletion of potentially self-reactive T cells (6, 7, 8), whereas expression of the Ag in peripheral (extrathymic) tissues often induces tolerance by nondeletional mechanisms, leading to clonal nonresponsiveness or anergy (9, 10, 11, 12, 13). In some settings with peripheral expression of the Ag, the function of anergic self-reactive T cells has been recoverable, suggesting that these cells could potentially be available for use in tumor therapy (12, 13, 14, 15). An alternative nondeletional outcome for CD8+ T cells that are specific for a peripherally expressed Ag has been defined as ignorance (16). Mice expressing an lymphocytic choriomeningitis virus (LCMV)-gp transgene in the pancreas showed no signs of autoimmunity and possessed the same T cell response to LCMV-gp after stimulation as wild-type mice. However, the T cells triggered by LCMV infection were no longer ignorant of the normal peripheral tissue and mediated rapid destruction of LCMV-gp-positive pancreatic islet cells leading to diabetes. These data suggest that a low level of a self-Ag in a noninflammatory milieu can be ignored by naive CTL, but raise the concern that activation of the T cells, such as by intentional immunization to a TAA, may alter the threshold for triggering and result in recognition of previously ignored normal tissues and autoimmune injury.
Studies in humans have demonstrated that T cells that recognize epitopes from normal proteins expressed in tumors, including both lineage-specific proteins and overexpressed cellular oncogenes, can be isolated in vitro (1, 17, 18, 19). However, this does not preclude in vivo anergy induction and deletion, as the recovered T cells may be of too low affinity to recognize either tumor or normal targets (20). Thus, models are necessary to define the nature of responsiveness to peripherally expressed TAA, determine the conditions by which nonresponsiveness can be overcome, and elucidate which categories of TAA can be safely and effectively used as targets for T cell therapy of tumors.
We describe here a C57BL/6 (B6)-transgenic (TG) mouse model in which the gag protein from the oncogenic Friend murine leukemia virus (FMuLV) is expressed under the control of the mouse albumin promoter in the liver. This FMuLVgag protein is also expressed by FBL, an FMuLV-induced erythroleukemia of B6 origin, and is the immunodominant target of the CD8+ T cell response in B6 mice to the tumor (21, 22). CD8+ T cells recognizing FMuLVgag can mediate complete eradication of disseminated leukemia following adoptive transfer into tumor-bearing B6 mice, so long as CD4+ T cell help or exogenous IL-2 is provided to sustain the CD8+ response (23, 24). By using the gag-TG mice, we describe a model for immunotherapy against a tolerizing self-tumor Ag and characterize the consequences of transferring tumor-reactive CD8+ T cells that also recognize the transgene as treatment of disseminated leukemia.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The construct CB16 containing the FMuLVgag sequence from FMuLV clone 57 has been described previously (25). In this construct, a CTG start site at nt 325 encoding glycosylated FMuLVgag and the ATG start site at nt 619 encoding gag have been mutated to prevent translation initiation, and the CTG site at nt 359 encoding glycogag mutated to ATG to optimize translational initiation from this site (25, 26). This construct was selected because the immunodominant CD8+ response to FMuLVgag in B6 mice is contained in the glycogag protein (22). An expression construct, pAlb-int-gag359, was generated in which the FMuLVgag sequence from CB16 was placed under the control of the albumin enhancer/promoter and the rat insulin intron A sequence inserted to improve expression (27). A construct, pAlb-gag359, was generated by the following sequential molecular manipulations: excision of a 2279-bp SpeI/SstI fragment from the CB16 construct, blunt-ending, and subcloning the fragment into the Alb-hgh vector (28) previously digested with BamHI and SmaI (to excise the hgh gene). Correct orientation of the gag sequence relative to the albumin promoter/enhancer was determined by restriction analysis. To generate Alb-int-gag359, a 150-bp rat insulin intron A fragment was excised from the plasmid pUC18/rat insulin II intron A using BamHI and BglII and ligated into the plasmid pAlb-gag359 that had been digested with BamHI. Correct orientation of the insert was determined by restriction analysis.
For injection into mice, pAlb-int-gag was digested with KpnI and ClaI to release the expression cassette from the vector backbone, electrophoresed onto a 1.2% agarose gel, and the sequence purified using QIAEX beads (Qiagen, Chatsworth, CA.). The DNA was resuspended in endotoxin-free dH2O, quantified, and injected into mouse embryos by using standard techniques. FMuLVgag-positive founder mice, as well as subsequent gag-positive offspring, were identified by analysis of tail DNA by using standard PCR protocols with FMuLVgag-specific primers (forward, 5'-GACTAGCTCTGTACCTGGCGGA-3'; reverse, 5'GATCTCTACCGACTGGTTGTGG-3').
Expression of FMuLVgag in gag-TG and control B6
mice was analyzed by semiquantitative RT-PCR of mRNA from the kidney,
liver, spleen, and thymus of adult mice. Tissue fragments were
snap-frozen on removal from mice, and mRNA was isolated and purified
from the tissue fragments as well as positive (4 x
106 in vitro FBL cells) and negative (water)
controls, with the Strataprep Total RNA Miniprep Kit (Stratagene, La
Jolla, CA). The matrix-bound, isolated mRNA was digested with DNase I
to eliminate potential contaminating DNA. Reverse transcription of the
purified mRNA was performed with the Thermoscript RT-PCR System (Life
Technologies, Grand Island, NY). First-strand synthesis of isolated
mRNA was achieved using oligo(dT)20 to prime
polyadenylated RNA. To verify the absence of contaminating DNA, the
reverse transcription reaction was also conducted in parallel samples
without reverse transcriptase. PCR amplification of 
-actin was
performed on both reverse transcriptase-treated and non-reverse
transcriptase-treated first-strand synthesis products, both to provide
a standard for the quality of the reverse transcription and to confirm
the absence of genomic DNA contamination. Actin was amplified using
Platinum Taq Polymerase (provided in the Thermoscript RT-PCR
System) and custom actin primers (forward, 5'-GTGGGCGCCCCAGGCACCA-3';
reverse, 5'-GTCCTTAATGTCACGCACGATTTC-3'). First-strand products were
normalized by titration of starting material based on the band
intensity of the amplified -actin. PCR amplification of
FMuLVgag was conducted on the normalized
first-strand products by using the custom
FMuLVgag primers. PCR products were then run out
on a 2% agarose gel. The PCR protocols for the amplification of
-actin and FMuLVgag were identical: 94°C for
1 min, 60°C for 1 min, and 72°C for 1 min for each of 34
cycles.
The presence of FMuLVgag protein in the liver of gag-TG mice was assessed from snap-frozen liver tissue that was lysed in lysis buffer, run on a 12% SDS-PAGE gel, and blotted to nitrocellulose filter. After blocking in 2% milk, the filters were probed with supernatant from the rat hybridoma R187 (a kind gift of B. Chesebro, Rocky Mountain Laboratories, Hamilton, MT (29)), washed, and specific binding detected by incubation with secondary HRP-conjugated anti-rat Ab and ECL Western blotting detection reagents (Amersham Pharmacia, Piscataway, NJ).
Cell Lines, Medium, and Peptides
FBL-3, a Friend virus-induced erythroleukemia of B6 (H-2b) origin, expresses FMuLV env- and gag-encoded products and MHC class I molecules but does not express MHC class II molecules (23). E10 is a FMuLV negative subline of the B6 EL-4 thymoma. The mapping and the sequence (CCLCLTVFL) of the dominant H-2Db restricted FMuLVgag epitope in FBL in B6 mice has been described (22), and the peptide was synthesized at the University of Washington (Seattle, WA). Unless otherwise stated, all cell culture was performed in RPMI 1640 supplemented with antibiotics, 2-ME, and 10% FCS.
Maintenance and immunization of mice
B6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME) The transgenic B6 mice were established and bred in our animal facility, and all mice were maintained under specific pathogen-free conditions. Six- to 8-wk-old mice were immunized twice with 5 x 106 irradiated FBL (10,000 rad) cells intraperitoneally (i.p.) 2 wk apart. Immune splenocytes were harvested 2–3 wk after the second immunization.
Adoptive chemoimmunotherapy (ACIT)
Adoptive immunotherapy experiments were performed as previously described (23). Briefly, on day 0, recipient mice were inoculated i.p. with 5 x 106 live FBL leukemia cells. On day 5, after the tumor cells have widely disseminated, mice were treated with 180 mg/kg cyclophosphamide (CY) i.p., followed in 6 h by adoptive transfer of splenic donor cells from immune mice, or of FBL-reactive CTL lines or clones grown and expanded in vitro, administered i.p. Splenocytes administered as effector cells were first depleted of CD4+ cells using Dynabeads (Dynal, Lake Success, NY) according to the manufacturer’s protocol, and the contaminating CD4 cells were always < 2% of the injected T cell population. T cells grown in vitro were analyzed by FACS and confirmed to be essentially 100% CD8+.
Analysis of autoimmune injury.
Groups of gag-TG and B6 mice treated with ACIT as above were sacrificed at 2–14 days after T cell transfer. Liver samples were fixed in formaldehyde, paraffin-embedded, sectioned, and stained with hematoxylin/eosin. Coded specimens were analyzed for inflammation and lymphocyte infiltration by a reference pathologist (Phoenix Central Laboratories, Everett, WA). Concurrent peripheral blood samples were analyzed for alanine aminotransferase and aspartate aminotransferase as biochemical indications of liver injury (Phoenix Central Laboratories).
Generation and testing of FBL-specific T cells
A total of 2.5 x 107 immune splenocytes from B6 or gag-TG mice were stimulated with 5 x 106 irradiated FBL cells for 5 days in T25 tissue culture flasks. After 5 days, the CTL were tested in standard 51Cr release assays against labeled FBL, E10 cells alone, or E10 pulsed with FMuLVgag peptide at the indicated concentrations. CTL lines were generated in vitro by repetitive stimulation of 2 x 106 cultured cells with 2 x 106 irradiated (10,000 rad) FBL in T25 flasks containing 5 x 106 irradiated (2,000 rad) B6 splenocytes as feeders and 20 U/ml IL-2 in a total volume of 10 ml. Lines were tested for cytolytic activity 5 days after an in vitro stimulation.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The single FMuLVgag open reading frame codes
for two alternative translational products:
Pr65gag and gPr75gag.
Transcription of the larger gag protein gPr75gag
is initiated from a CTG start site at position 355 of the FMuLV
sequence, which is upstream and in-frame with the ATG start site for
the conventional gag protein Pr65gag at nt 619.
The higher m.w. protein, which has been named glycogag, has an
additional 98-aa N-terminal leader sequence preceding the amino acid
sequence of Pr65gag (25, 26) Because
translation is predominantly initiated at the ATG start site at nt 619,
the major protein species generated from this open reading frame is
Pr65gag (25). However, the dominant
CTL epitope elicited in B6 mice in response to
FMuLVgag is located in the N-terminal leader
segment of the FMuLV gPr75gag protein, encoded by
FMuLV nt 577–603 (22). Therefore, the gag transgene was
designed to optimize transcription and expression of this
immunodominant CTL epitope. An FMuLVgag gene was
constructed (kindly provided by Dr. Jean-Luc Darlix, Lyon, France) in
which the CTG start site for gPr75gag at nt 355
was mutated to an ATG, and the ATG start site for
Pr65gag at nt 619 mutated to an AGC (Fig. 1
A). In this construct,
translation has been demonstrated to occur almost exclusively from nt
355 (25). The construct was subcloned into a vector
optimized for selective expression in the liver of transgenic mice off
an albumin promoter (kindly provided by Dr. Richard Palmiter, Seattle,
WA) (28) (Fig. 1B). The construct was
microinjected into pronuclei of fertilized eggs from B6 mice according
to standard protocols. A founder positive for the transgene was
identified by PCR of tail DNA (data not shown), which transmitted the
transgene to subsequent generations.
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top panel), with the amount
of cDNA normalized to -actin as control (Fig. 2 lower
panel). A robust band of the expected size (417 bp) was amplified
from cDNA generated from liver tissue of gag-TG mice, whereas cDNA from
the liver of nontransgenic littermates was negative. Considerably
weaker bands also were detected in other tissues from gag-TG after 34
cycles of PCR, suggesting some leakiness of the promoter. These bands
did not appear to result from potentially contaminating DNA, as
amplification of RNA samples mock treated without reverse transcriptase
failed to generate a signal (data not shown). No
FMuLVgag mRNA expression was detected in tissues
from B6 mice.
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). The lane containing the FBL tumor
lysate had a strong band of 65 kDa size as well as a weak band at 
75
kDa, presumably reflecting both the Pr65gag and
gPr75 gag. Neither band was detectable in lysates
from normal B6 mice. The gag-TG liver lysate had a single band at 75
kDa, consistent with the modification of the transgenic construct to
translate only the larger protein species.
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To investigate the immunological consequences of expressing
FMuLVgag as a TAA, gag-TG and normal B6 mice were
primed and boosted in vivo with irradiated FBL. Two weeks after the
second immunization, spleens were harvested, stimulated in vitro with
irradiated FBL, and tested for the presence of FBL-reactive CTL. After
in vitro sensitization for 5 days, spleen cells from in vivo-primed
normal B6 mice exhibited specific lytic activity for FBL (Fig. 4). This CD8+ T
cell mediated (Ref. (21) and data not shown) cytotoxic
activity recognized the dominant CTL epitope, as demonstrated by
pulsing FMuLV- negative targets with the peptide defining this gag
epitope. In contrast, no CTL activity to FBL or to targets pulsed with
the immunodominant epitope could be detected in cultures derived from
gag-TG mice. Thus, the gag-TG mice are not only tolerant to
FMuLVgag, but also fail to generate CTL responses
to any potentially subdominant MHC class I-restricted epitopes encoded
by other genes in FBL.
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In a clinical setting, adoptive therapy will require a means to recover and expand potentially tolerant T cells reactive with the TAA. The failure to detect a CTL response in gag-TG animals could be the result of anergy, from which recovery would potentially be possible, or from permanent deletion of the FMuLVgag-specific repertoire. Therefore, we attempted to overcome tolerance and expand FMuLV-reactive CD8+ T cells by removing the cells from the tolerogenic in vivo environment and repetitively stimulating the cells in vitro in the presence of supplemental IL-2.
Mice were primed and boosted in vivo with irradiated FBL, and
their splenocytes then stimulated weekly in vitro with irradiated FBL
and IL-2. Cell cultures from primed B6 mice expanded with each
stimulation cycle, but the cultures from gag-TG mice exhibited an
initial contraction, followed by stable cell numbers, and then
proliferation after three cycles. Analysis of cytolytic activity after
the fourth cycle revealed FBL-reactive CTL in T cell lines from gag-TG
animals that killed FBL targets similar to CTL lines from normal B6
animals (Fig. 5). No lytic activity was
detected from similarly in vitro-stimulated T cells derived from naive
B6 mice (data not shown), suggesting that this method does not
effectively prime naive CD8+ T cells in vitro.
The specificity of the CTL for gag and their relative affinity was
assessed by titrating the immunodominant peptide epitope on Ag-negative
E10 targets. Only small differences in lytic activity by CTL from
gag-TG and normal B6 mice were observed over peptide titrations from
0.01 to 10 µg/ml (Fig. 5). Repeat experiments with additional CTL
lines at ranging peptide doses suggested no reproducible differences
between the CTL lines. Although other factors could contribute to the
avidity of these CTL lines for the target, the peptide titration
studies suggest that the CTL derived from gag-TG mice are of relatively
similar affinity to those from B6 mice. These results suggest that the
CTL recovered from the tolerant mice have a repertoire containing some
high-affinity and not just residual low-affinity T cells. The recovery
of such higher-affinity T cells after repetitive in vitro stimulation
likely reflects the reversal of anergy in tolerant cells in this
environment rather than just outgrowth of rare T cells that have not
undergone deletion, as repetitive in vivo stimulations in the tolerant
mice failed to result in detectable expansion of gag-reactive T cells
(data not shown).
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).
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Even if T cells reactive with a TAA can be rescued as above, such
T cells might still not be useful for therapy. First, the mechanisms
operative for maintaining tolerance in gag-TG mice could potentially
similarly induce anergy in transferred T cells specific for the TAA and
interfere with efficacy. Second, if the tolerizing signals prove
incapable of anergizing infused effector T cells, these CTL, which are
targeted to a protein shared between the tumor and normal tissues,
could potentially lead to severe autoimmunity. Therefore, B6 and gag-TG
mice were inoculated with FBL tumor cells on day 0, and received
treatment on day 5 with cyclophosphamide followed in 6 h by
transfer of 107
FMuLVgag-specific CTL derived from a B6 mouse.
The mice then received on days 5–19 104 U
IL-2/day i.p. FBL tumor was eradicated by this treatment regimen in
both the gag-TG and B6 mice (Fig. 7).
Thus, the FMuLVgag expressed in the liver did not
interfere with the in vivo antitumor activity or tolerize primed
activated CTL, at least during the 4-wk time required for tumor
eradication (30). The results suggest that primed T cells
have a different threshold for tolerance induction than naive T
cells.
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) nor measurement of AST and ALT serum
levels (data not shown) at any time point revealed evidence of
autoimmune injury in treated gag-TG animals.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A major obstacle to pursuing T cell therapy targeting tumor Ags that also are expressed in normal tissues is the difficulty in isolating such T cells, which is at least in part attributable to tolerance in the host. This obstacle was evident in the gag-TG mice, as initial efforts to immunize the mice with FMuLVgag in vivo to expand the number of gag-specific CTL were unsuccessful. However, FMuLVgag-reactive T cells with efficacy in tumor therapy similar to nontolerant T cells from immunized B6 mice could be recovered from gag-TG mice after removal from the in vivo tolerizing environment. The approach employed, involving repeated cycles of in vitro stimulation in combination with supplemental IL-2, is analogous to methods we have used to isolate human melanoma-reactive T cells specific for normal melanocyte Ags from patients with melanoma (31).
Other studies that used nominal Ags have also suggested that maintenance of the anergic state in self-reactive T cells requires continuous exposure of the T cells to the tolerogen, and that the nonresponsive state can be reversed after withdrawal of the tolerogen. (15, 32, 33). However, the persisting recovered T cells may have a lower affinity than T cells isolated from hosts not expressing the Ag. In a transgenic model in which hemagglutinin from influenza virus was expressed in islet cells in the pancreas, the recovered T cells recognized a transfected renal carcinoma cell line despite exhibiting only low affinity for hemagglutinin (34). These CD8+ T cells were capable of rejecting the tumor in vivo without causing detectable autoimmune injury. Thus, nontolerant low-affinity T cells reactive with TAA may be isolated in settings in which high-affinity T cells have been tolerized and may be effective in vivo without inducing toxicity if the tumor expresses a sufficient amount of Ag to be recognized. However, our results suggest that higher-affinity T cells might also be recoverable in some settings because peptide titrations provided inferential evidence in our model that the recovered TAA-reactive T cells were of similar affinity to those detected from normal immunized hosts.
Another potential obstacle for therapy with tumor-reactive T cells that also recognize normal tissues is that the infused T cells might be tolerized after transfer and fail to mediate antitumor activity. However, the therapeutic efficacy observed after transfer of FMuLVgag-reactive primed T cells suggest that the requirement for tolerizing primed T cells is distinct from the requirements for tolerizing naive T cells. This appears to be the reciprocal of the requirements for activation of primed T cells, which requires lower levels of Ag and is less dependent on costimulatory and accessory molecules than activation of naive T cells (35, 36). Thus, a tolerizing signal for naive T cells may be perceived as a competent activating signal by primed T cells.
If the tolerizing peripheral tissue can actually activate primed but not naive T cells, then transfer of primed cells would be expected to mediate acute and/or chronic injury to the normal tissue expressing the proteins. However, histological and biochemical analysis of liver tissue from gag-TG mice that received immune T cells that were capable of rejecting a tumor challenge, an activity that requires retention of function for at least 30 days (30), failed to reveal evidence of liver injury. Thus, despite expression of FMuLVgag in the liver adequate to induce nonresponsiveness in naive T cells, activated effector cells did not promote injury to normal tissue. This may reflect differential susceptibilities of normal tissues and the tumor to the cytotoxic effector mechanisms and/or be a consequence of quantitatively different levels of Ag expression. Other murine models have also suggested that normal tissues may be more resistant to injury than tumor cells (34, 37, 38). We previously reported that transgenic mice expressing from an Ig promoter the FMuLV-env gene, which is the target for an MHC class II-restricted FBL-specific CD4+ T cell response, were resistant to autoimmune injury despite the ability of adoptively transferred T cells to reject tumor in these mice (39). In that model, however, both the tumor and some self tissues (i.e., T cells) expressing the Ag are MHC class II negative, requiring indirect effector mechanisms to mediate the antitumor activity as well as selected autoimmune injury. In contrast, in the current model both tumor and hepatocytes are MHC class I positive and therefore potentially directly sensitive to the lytic activity of the CD8+ CTL used in ACIT. In the gag-TG mice, liver parenchymal cells may partially resist injury attributable to expression of limited amounts of the counterreceptors, such as CD80 or CD86, that contribute to T cell-target avidity and provide costimulatory signals (40). Additionally, for some targets such as EBV-infected cells, susceptibility to the effector phase of the CD8+ response requires a higher density of the relevant viral Ags than stimulation of CD8+ responses (41).
Induction of tolerance can occur if APC take up self-Ag and then present it to T cells in a draining lymph node. This is analogous to some of the events involved in cross-priming, but presumably reflects failure to activate the APC with resultant delivery of tolerizing rather than activating signals (42, 43). Thus, the gag Ag may not be directly presented to T cells by hepatocytes, but rather by professional APC such as dendritic cells. Uptake of released Ag from uninjured liver cells or phagocytosis of apoptotic liver cells might not be expected to promote dendritic cell maturation. However, we attempted to promote autoreactivity by inducing inflammatory responses in the liver, followed by transfer of FMuLVgag-specific CTL. These experiments included infection of the livers of gag-TG mice with vaccinia or adenovirus, followed by injections of irradiated FBL as a stimulating Ag source, and IL-2 to promote survival and proliferation of the transferred CTL. No evidence of injury to the liver was detectable following resolution of the acute infections (data not shown). This resistance of hepatocytes to autoimmune damage may be a general phenomenon. In a transgenic model in which tolerance was induced by expression of the alloantigen H-2Kb in the liver, hepatocytes were very resistant to autoimmune injury (44), although CTL-mediated liver damage could be induced by infecting the mice with the liver-specific pathogen Listeria monocytogenes. The ability to ultimately induce autoimmune injury in this setting may be due to the tolerizing Ag being an allogeneic class I Ag, which cannot induce tolerance by cross-presentation via host APC and should be recognized by a broader host response.
Additional evidence suggesting liver cells may be insensitive targets
for killing by T cells has been provided by studies of viral hepatitis.
CD8+ T cells controlled viral replication in
infected hepatocytes by release of inflammatory cytokines such as
IFN-
and TNF (45). Surprisingly, this biological
activity was not accompanied by a cytopathic effect on hepatocytes.
Thus, CD8+ T cells capable of killing targets can
recognize liver cells and perform effector functions while not lysing
the liver cells. The means by which hepatocytes may be protected from
cytolysis are unclear, but similar mechanisms could be operative in our
model.
The results of this murine study are consistent with observations following T cell therapy for human melanoma, in which T cells reactive with Ags expressed in both melanoma tumor cells and human melanocytes have been found to be therapeutically effective without mediating toxicity in most patients and inducing partial vitiligo in the remaining fraction (46). Our data validate the hypothesis that therapies based on T cell immunity to TAA overexpressed in tumor cells have the potential to provide significant therapeutic benefit. The ability to isolate and use such tumor-reactive T cells will likely be strongly dependent on the particular tissues that express the self-Ag, as well as the magnitude of normal expression. Further studies in relevant murine models such as the one presented here should help elucidate the principles necessary to facilitate development of this approach for human tumor therapy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Claes Öhlén, Department of Immunology, Box 356527, University of Washington, Seattle, WA 98195.
3 Current address: Corixa Corporation, 1124 Columbia Street, Seattle, WA 98104.
4 Abbreviations used in this paper: TAA, tumor-associated Ags; LCMV, lymphocytic choriomeningitis virus; B6, C57BL/6; FMuLV, Friend murine leukemia virus; FBL, an FMuLV-induced erythroleukemia of B6 origin; TG, transgenic; ACIT, adoptive chemoimmunotherapy; CY, cyclophosphamide.
Received for publication August 24, 2000. Accepted for publication November 27, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C. Ohlen, M. Kalos, L. E. Cheng, A. C. Shur, D. J. Hong, B. D. Carson, N. C.T. Kokot, C. G. Lerner, B. D. Sather, E. S. Huseby, et al. CD8+ T Cell Tolerance to a Tumor-associated Antigen Is Maintained at the Level of Expansion Rather than Effector Function J. Exp. Med., June 3, 2002; 195(11): 1407 - 1418. [Abstract] [Full Text] [PDF]
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