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Department of Medicine, Division of Oncology, Stanford University School of Medicine, Stanford, CA 94305; and
Division of Hematology and Oncology, Research Service 11R, Veterans Administration Medical Center, Ann Arbor, MI 48105
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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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Given the encouraging results of B cell Id vaccines in the treatment of B cell malignancies and the similarities between B and T cell Ag receptors, we have established and characterized a murine T cell lymphoma model system for the development of TCR vaccines (8). The use of TCR as immunogen, especially the use of V region peptides, has been well documented in the treatment of autoimmune diseases (9, 10, 11, 12). However, the immune response induced by V region peptide therapy resulted in the deletion or suppression of normal T cells in addition to self-reactive T cells having the same V regions (13, 14, 15), which is undesirable in antitumor immunotherapy. To develop an antitumor vaccine that would induce an immune response specific for tumorigenic T cells without resulting in the deletion of normal T cells, we reported the use of soluble, heterodimeric TCR as immunogens in vaccines for active tumor immunotherapy that was Id specific (8). One of the major obstacles in evaluating the efficacy of tumor-specific TCR as an active immunotherapy vaccine was the production of sufficient quantities of soluble, heterodimeric TCR proteins. Since TCR proteins are not secreted, we have cloned and overexpressed a recombinant TCR derived from the murine T cell tumor C6VL as a phophatidylinositol-linked (PI)3 protein on transfected cells (8, 16). After digestion with PI-specific phospholipase, large quantities of soluble C6VL TCR proteins were obtained and purified. In our previous study, we demonstrated the antitumor effects of TCR vaccine in active immunotherapy of the murine T cell tumor C6VL. Mice immunized with soluble C6VL TCR conjugated to keyhole limpet hemocyanin (KLH) in the presence of syntex adjuvant Formulation-1 (SAF-1) induced a strong C6VL TCR-specific humoral immune response. TCR-vaccinated mice were protected against a lethal dose of C6VL tumor cells. No deleterious effect was observed on normal T cell populations, nor skewing of the TCR repertoire, and the protection was specific against C6VL and not other T cell tumors.
In this study, we have characterized the immune mechanisms involved in tumor protection of vaccinated mice. Different immunologic adjuvants were used in an attempt to improve the efficacy of TCR vaccines, and to understand the correlation between tumor protection and the quality of immune response induced. We have found that adjuvants that bias the immune response toward Th1-type, reflected by the induction of IgG2 Abs and effector CD8+ T cells, confer the best protection. We have further found that CD8+ T cells are necessary and sufficient for tumor protection in this model system, and that anti-C6VL TCR Abs provide only mild antitumor effects. Understanding of the mechanisms of tumor protection induced by the TCR vaccines will facilitate the design of future immunotherapies for T cell malignancies.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 mice (H-2b) (age 6–8 wk) were purchased from Charles River Laboratories, Wilmington, MA. B cell-deficient mice (JHD) (17) were obtained from GenPharm (Mountain View, CA), and backcrossed onto the C57BL/6 background (kindly provided by D. Umetsu, Stanford, CA). All mice were housed at the Laboratory Animal Facility at Stanford University Medical Center (Stanford, CA). C6VL (H-2b) is a murine tumor cell line derived from a radiation-induced thymoma of C57BL/Ka background (18). Clone 1D6-480-4 is a transfected BW5147.G.1.4 cell line that overexpresses C6VL TCR as PI-linked proteins on the cell surface (8, 19). Rat hybridoma 2.43 (rat IgG2b anti-mouse CD8.2) was obtained from the American Type Culture Collection (Rockville, MD). Rat hybridoma SFR8-B6 (rat IgG2b anti-human HLA Bw6) was kindly provided by J. Parnes, Stanford, CA.
C6VL TCR purification
Recombinant C6VL 
ß-TCR was obtained as previously described
(8). Briefly, C6VL TCR and ß genes were isolated and
then modified such that the transmembrane regions of the C regions were
replaced with sequences from 3' region of the decay-accelerating factor
(DAF) gene, which encodes for a PI linkage domain (20).
BW5147 cells transfected with the modified C6VL ß-TCR/DAF genes
expressed C6VL TCR as a PI-linked protein on the cell surface. The C6VL
TCR/DAF expression was amplified with methotrexate selection. A high
TCR-expressing clone (1D6-480-4) was used for the source of the
recombinant C6VL TCR. TCR protein was released from the cell surface by
incubating the TCR transfectants with PI-phospholipase C (PLC), a
phospholipase specific for the PI linkage. Soluble C6VL ß-TCR was
purified from the PI-PLC-digested supernatants with a H57-597 (mAb
anti-Cß) Sepharose affinity column. Affinity-purified TCR protein
was concentrated using Con A lectin Sepharose affinity column (Sigma
Chemical Company, St. Louis, MO). Purified C6VL ß-TCR was dialyzed
extensively against PBS and then filter sterilized. TCR concentration
was determined by BCA protein assay (Pierce Chemical Co.,
Rockford, IL).
TCR immunizations
Purified C6V1 ß-TCR were chemically conjugated to KLH
(Calbiochem, San Diego, CA) at 1:1 ratio (w/w) using glutaraldehyde as
previously described (2). Mice were immunized s.c. with
TCR-KLH conjugates containing 35 µg (500 pmol) TCR in various
adjuvant formulations in a total volume of 200 µl PBS. SAF-1 was
prepared as previously described (2, 21). SAF-1 is
composed of 0.2% v/v Tween-80, 5% v/v squalene, 2.5% v/v Pluronic
L121, and 100 µg/ml Thr1-MDP. Thr1-MDP was
included only in the first immunization. QS-21 (kindly provided by
Aquila Biopharmaceuticals, Framingham, MA) is a purified saponin-based
adjuvant from Quillaja saponaria Molina extracts (22, 23), and was given at 10 µg per injection. Murine rIL-12
(kindly provided by Stan Wolf, Genetics Institute, Cambridge, MA) was
given at 0.5 µg per injection. Both recombinant murine
granulocyte/macrophage CSF (GM-CSF) and soluble trimeric murine CD40
ligand (CD40L) were kindly provided by Immunex Corporation (Seattle,
WA). CD40L was given at 2.5 µg per injection. GM-CSF was given in
four 1-µg doses per immunization, with 1 µg GM-CSF coinjected with
TCR-KLH, followed by three 1-µg doses given 1, 2, and 3 days after
TCR immunization (24). The irrelevant protein vaccine
control consisted of Id protein derived from the murine B cell lymphoma
38C13, similarly conjugated to KLH and given in equal molar amount in
adjuvants (2). Immunizations were given three times at
2-wk intervals. Serum samples were collected 10 days after each
immunization.
Anti-C6VL ß-TCR ELISA assay
ELISA assays were performed as previously described
(8). Briefly, 96-well Maxisorb plates (Nunc, Naperville,
IL) were coated with anti-Cß mAb H57-597. Purified C6VL
ß-TCR diluted in PBS containing 2% BSA was bound to
H57-597-coated plates. Mouse immune serum was diluted and titered over
eight wells in twofold dilutions. A standard curve was generated by
titering mAb 124-40, a mouse IgG anti-C6VL TCR clonotype encoded on
-chain V region (18). Bound mouse Abs were detected
using a peroxidase-conjugated donkey anti-mouse Ig Ab (Jackson
ImmunoResearch, West Grove, PA). After removal of unbound proteins,
substrate solution containing 2,2'-azinobis(3-ethyl-benzthiazoline
sulfonic acid (ABTS) was added to each well. The color reaction was
allowed to develop at room temperature. Absorbance at 405-450 mm was
measured using a Vmax microplate reader
(Molecular Devices, Menlo Park, CA). Anti-C6VL ß-TCR Ab titers
were determined from the linear portion of the standard curve.
Affinity elution with ammonium thiocyanate
ELISA assays were performed as described above with the
following modification (25, 26). Ninety-six-well
microtiter plates were coated with 2 µg/ml purified C6VL ß-TCR
in 50 mM carbonate buffer (pH 9.0). Pooled immune serum from groups of
10 mice each vaccinated with C6VL ß-TCR in various adjuvants was
incubated on the TCR-coated plates for 1 h at room temperature at
0.1 µg/ml anti-C6VL ß-TCR Ab (titers were determined from
previous ELISA assays). The Ab concentration was chosen such that the
absorbance readings were near the top of the linear portion on the
standard curves, and were equivalent among different adjuvant groups.
After washing, 100 µl of ammonium thiocyanate (NH4SCN) in
PBS ranging from 0 to 3 M was added to replicate wells. The plates were
incubated for 15 min at room temperature and washed. Bound Abs were
detected using a peroxidase-conjugated donkey anti-mouse Ig Ab. The
absorbance reading in the presence of 0 M NH4SCN was
defined as 100% initial binding. Affinity index was defined as
the molar concentration of thiocyanate required to reduce the initial
absorbance reading by 50% (25, 26).
Isotype-specific ELISA
ELISA assays were performed as described above with the following modification. Pooled serum from groups of 10 mice each were captured on TCR-coated plates. After 1-h incubation, bound mouse Abs were detected by using peroxidase-conjugated goat anti-mouse IgG1, IgG2a, IgG2b, and IgG3 Abs (Southern Biotechnology Associates, Birmingham, AL). Relative units were calculated using a pooled serum standard, with the starting dilution of the standard arbitrarily set as 1. All serum samples were calibrated similarly against the serum standard.
Tumor challenge
Two weeks after the third protein immunization, mice were challenged with a lethal dose of C6VL cells. A frozen aliquot of C6VL cells was thawed and was grown in RPMI 1640 containing 10% FCS and 50 µm 2-ME for 3 days. The tumor cells were collected, washed three times in HBSS, and diluted to 1 x 104 cells/ml. Mice were injected i.p. with 5000 C6VL tumor cells in 500 µl HBSS. Survival of mice challenged with tumor was monitored for at least 60 days after tumor injection. Survival curves were generated using the Kaplan-Meier method. Statistical analysis was done using the log-rank test.
Transfer of hyperimmune serum
Hyperimmune serum was obtained from 40 C57BL/6 mice immunized
three times with C6VL ß-TCR-KLH in QS-21. Serum was collected and
pooled. The hyperimmune globulin fraction was precipitated by adding a
saturated ammonium sulfate solution slowly to the serum to a final
concentration of 45% v/v, and incubated overnight at 4°C.
Precipitated proteins were pelleted by centrifugation at 20,000 x
g for 30 min at 4°C. The protein pellet was resuspended in
a minimal amount of PBS and dialyzed extensively in PBS. The proteins
were further concentrated using Centriplus-30 concentrators (Amicon,
Beverly, MA). Final anti-TCR Ab titers as well as total IgG
concentrations were determined by ELISA as described above. Recipient
mice were injected i.p. with 500 µl concentrated hyperimmune globulin
protein. One day after serum transfer, mice were challenged with C6VL
tumor cells and followed for survival. For negative Ab control, serum
from either naïve mice or mice vaccinated three times with
38C13 Id-KLH in QS-21 was collected and similarly concentrated. Control
mice were injected with 500 µl concentrated irrelevant globulin
protein containing equivalent amount of IgG as compared with the
anti-TCR hyperimmune globulin protein. Mice were bled 2 h
before tumor challenge and the level of circulating anti-TCR Ab
titer was determined by ELISA.
In vivo depletion of CD8+ T cells
C57BL/6 mice were depleted of CD8+ T cells using the anti-CD8 mAb 2.43 (rat IgG2b). Control mice were injected with an irrelevant isotype-matched mAb SFR8-B6 (anti-human HLA Bw6). Both 2.43 and SFR8-B6 hybridomas were grown as ascites in pristane-primed nude mice, mAbs were harvested as ascitic fluid, diluted in PBS, and filter sterilized. Concentration of mAbs was determined using a rat IgG2b-specific ELISA assay. Mice were injected i.p. with 250 µg mAb in 500 µl PBS on days 6, 5, and 4 before tumor challenge. Three weekly injections were given post-tumor challenge to maintain the depletion, starting 1 wk after the third mAb injection. The extent of CD8+ T cell depletion in peripheral blood was analyzed by flow cytometry 1 day before tumor challenge and 3 days after the last weekly mAb treatment (22 days post-tumor challenge) using a nonblocking anti-CD8 mAb (PharMingen, San Diego, CA).
Adoptive transfer of T cell-enriched lymphocytes
Fifteen C57BL/6 donor mice were immunized three times with C6VL
ß-TCR-KLH in QS-21 as described above. Control donor mice were
similarly immunized with 38C13 Id-KLH in QS-21. Ten days after the last
immunization, donor mice were sacrificed to harvest spleens and
inguinal lymph nodes. Immune lymphocytes from donors were pooled. The
lymphocyte preparation was depleted of contaminating RBCs by
resuspending the cells in 0.144 M NH4Cl, 0.017 M Tris-HCl,
pH 7.2. To remove B cells, lymphocytes were resuspended in ice-cold
PBS/2% FCS and added onto sterile 15-cm petri dishes that were
previously coated with 125 µg goat anti-mouse Ig Ab (BioSource
International, Camarillo, CA) and 500 µg irrelevant Ig
(anti-human Id mAb LC4) diluted in PBS. After incubation for 1
h at 4°C, the plates were gently swirled and nonadherent cells were
collected. The panning procedure was repeated once, and the B
cell-depleted lymphocytes were pooled from each plate, washed twice in
HBSS, and used for adoptive transfer. Lymphocytes were analyzed pre-
and post-B cell panning by flow cytometry to determine B and T
lymphocyte proportion, as determined by CD3+ and
CD19+ cell populations. Twenty million T cell-enriched
immune lymphocytes were injected i.v. in 200 µl HBSS into
naïve recipients which were exposed to a sublethal dose of 400
rad of whole body gamma irradiation (RT250 X-ray Unit, Philips Medical
Systems, Scarborough, Canada). Mice were challenged with C6VL tumor
cells 4 days after cell transfer.
Proliferation assay against KLH
Four days after adoptive transfer of lymphocytes, one recipient mouse from each adoptive transfer group not used in tumor challenge was sacrificed. Spleens were harvested and made into single cell suspension and depleted of RBCs as described earlier. Splenocytes were washed and resuspended in serum-free AIM-V medium (Life Technologies, Grand Island, NY) supplemented with 50 µM 2-ME. Fifty thousand cells were seeded per well in 96-well U-bottom tissue culture plates. KLH was added into each well at a final concentration of 100, 10, and 1 µg/ml and the cells were cultured at 37°C in a humidified incubator for 4 days. On day 3, 1 µCi of [3H]thymidine was added to each well. Cells were harvested onto glass fiber filters and counted on a scintillation counter (Wallac Micro Beta 1450, Turku, Finland).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether the efficacy of TCR vaccines could be
improved beyond what was previously reported (8), mice
were immunized with TCR vaccines formulated with various adjuvants. The
magnitude and quality of humoral immune response stimulated by TCR
vaccines composed of SAF-1, QS-21, IL-12, CD40L, or GM-CSF were
compared. After three vaccinations, serum samples were collected and
analyzed by ELISA for anti-C6VL TCR Ab titers. Control mice were
immunized with an irrelevant protein 38C13 Id-KLH given in matching
adjuvants. Mice immunized with TCR-KLH in QS-21 and SAF-1 were very
effective in inducing high amounts of anti-C6VL TCR Abs, with
average titers of 499 µg/ml and 329 µg/ml, respectively. In
contrast, mice immunized with TCR-KLH in CD40L, GM-CSF, and IL-12 had
5- to 10-fold lower Ab titers, with an average of 32 µg/ml, 86
µg/ml, and 46 µg/ml, respectively (Fig. 1
). The Ab were specific for C6VL TCR, as
serum from mice immunized with an irrelevant protein in matching
adjuvant did not make any anti-C6VL Ab response (data not
shown).
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). The levels of mouse
anti-C6VL TCR IgG1, IgG2a, IgG2b, and IgG3 present in hyperimmune
serum stimulated by different adjuvants were measured using a pooled
hyperimmune serum standard. Distinct Ab isotype profiles were produced
by the different adjuvants. IgG1 Abs were induced by all five adjuvants
tested. IgG2a Abs were detected only in mice immunized with IL-12 and
QS-21. IgG2b Abs were induced by IL-12, QS-21, and SAF-1. In contrast,
no IgG2a or IgG2b Abs were detected in the CD40L and GM-CSF groups.
IgG3 Abs were not detected in any of the serum samples tested. Data
shown are representative of at least two independent experiments. Each
IgG subclass required the use of a different isotype-specific Ab
detectors, thus the absolute quantities of IgG1, IgG2a, and IgG2b
within each adjuvant group may not be reflected by the relative units
measured in isotype-specific ELISAs.
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). Antisera induced by the difference
adjuvants were adjusted to equal anti-C6VL TCR Ab concentrations
and captured on TCR-coated ELISA plates. Bound Abs were exposed to
increasing concentrations of NH4SCN. Disruption of Ag-Ab
binding by chaotropic ions has been reported to correlate well with the
affinity of mAbs (25), and has been used to determine the
avidity of polyclonal Ab as well (26, 27). Resistance to
thiocyanate elution was used to determine relative Ab avidity in this
assay and compared with the mouse anti-C6VL clonotypic mAb 124-40.
The avidity among the different adjuvant groups were not significantly
different compared with the mAb 124-40, as there was an approximately
10% variation in interplate absorbance reading (25, 26).
In addition, the dissociation rate of bound anti-C6VL TCR Abs from
C6VL TCR was measured using the surface plasmon resonance biosensor. No
significant difference was detected in the rates of Ab dissociation
among the different adjuvant groups (data not shown).
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To test whether the immune response induced by TCR vaccines in
various adjuvants would inhibit tumor growth, we challenge mice
vaccinated with TCR vaccines in various adjuvants with a lethal dose of
C6VL cells. Only mice immunized with TCR vaccines given in SAF-1,
QS-21, and IL-12 were significantly protected against C6VL tumor
compared with PBS control (Fig. 3), with
p values of 0.01, 0.003, and 0.009, respectively. Mice given
TCR vaccines in CD40L and GM-CSF were not protected and died at rates
similar to control mice given PBS. The three protected groups were not
statistically different from one another. To rule out a nonspecific
protective effect due to adjuvants, additional mice were vaccinated in
subsequent experiments with an irrelevant protein, 38C13 Id-KLH, in
SAF-1, QS-21, and IL-12 and challenged with C6VL cells. TCR-vaccinated
mice had significantly prolonged median survival (>60 days) compared
with mice vaccinated with control protein (median survival of 
30
days) (Table II). Survival of control
mice was not statistically different from mice injected with PBS only.
The tumor protective effect of TCR vaccines in SAF-1, QS-21, and IL-12
adjuvants had been confirmed in at least three independent experiments.
The level of tumor protection by TCR vaccines would varying different
experiments, but statistical significance was always reached
(p < 0.05) when compared with negative control
groups.
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The antitumor effect of anti-C6VL TCR Abs was tested by
injecting naïve mice with a concentrated globulin fraction of
hyperimmune serum obtained from C57BL/6 mice vaccinated with TCR-KLH in
QS-21. The average anti-TCR Ab titer in recipient mice 1 day after
globulin transfer was 231 ± 25 µg/ml, comparable with actively
vaccinated mice, which had an average anti-C6VL TCR Ab titer of
247 ± 54 µg/ml (data not shown). Negative control mice were
injected with an equivalent amount of similarly prepared globulin
obtained from mice vaccinated with 38C13 Id-KLH in QS-21. One day after
the Ab transfer, mice were challenged with C6VL cells and followed for
survival. Mice transferred with anti-C6VL TCR hyperimmune globulin
had significantly prolonged survival compared with mice given control
globulin (Fig. 4). However, tumor
protection was not restored to the level achieved in actively
vaccinated mice.
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). The loss of tumor protection in
JHD mice could be due either to the absence of Abs, or to a
deficiency in B cell Ag presentation. We tested whether tumor
protection can be restored by transferring hyperimmune globulin
(obtained from C57BL/6 mice vaccinated with TCR-KLH in QS-21) into
JHD mice that were actively vaccinated with TCR-KLH in
QS-21. The anti-tumor effect of Abs alone was evaluated by
transferring hyperimmune globulin into JHD mice vaccinated
with 38C13 Id-KLH in QS-21. The average anti-TCR Ab titer in
recipient JHD mice 1 day after Ab transfer was similar to
the Ab titers obtained in C57BL/6 mice in Fig. 4
. Negative control
JHD mice were vaccinated with TCR-KLH in QS-21, and
injected with control globulin obtained from naïve C57BL/6
mice. One day after serum transfer, mice were challenged with C6VL
tumor. JHD mice given anti-C6VL TCR hyperimmune
globulin had prolonged survival compared with mice given control
globulin (Fig. 6). However, the degree of
tumor protection in mice first vaccinated with TCR-KLH and then given
anti-TCR Abs was not significantly different from mice that were
injected with anti-TCR Abs alone.
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The importance of T cell-mediated immune response in protection
against C6VL tumor in vivo was determined by two different approaches.
The role of effector CD8+ T cells in conferring tumor
protection was assessed in the first approach by using an anti-CD8
mAb to deplete CD8+ T cells in mice immunized with TCR-KLH
in QS-21. Mice were treated with either an anti-CD8 mAb, or an
isotype-matched mAb after the third vaccination and prior to tumor
challenge. The absence of CD8+ T cells in peripheral blood
was confirmed by flow cytometry using a non-cross-blocking anti-CD8
Ab. In the anti-CD8 mAb-treated group, greater than 98% of
CD8+ T cells were depleted (data not shown), and depletion
was maintained throughout the observation period with weekly injection
of depleting mAbs. The isotype matched irrelevant mAb had no effect on
CD8+ T cells population. TCR-vaccinated mice that were not
depleted of CD8+ T cells were significantly protected
against C6VL tumor as compared to mice vaccinated with PBS or a control
protein. Depletion of CD8+ T cells completely abrogated
tumor protection in QS-21 vaccinated mice (Fig. 7). Anti-C6VL TCR Abs were present in
both CD8+T cell-depleted and nondepleted groups, with an
average anti-C6VL TCR Ab titer of 235 ± 181 µg/ml, and
318 ± 192 µg/ml, respectively. Similar result was observed when
mice immunized with TCR vaccine in IL-12 were depleted of
CD8+ T cells (data not shown). The importance of
CD4+ T cells cannot be tested in this experiment, as
anti-CD4 Abs would bind to the CD4+ C6VL tumor cells in
addition to normal CD4+ T cells.
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Both adoptive transfer recipient groups had comparable KLH-specific
proliferative responses, indicating transfer of equivalent numbers of
immune competent cells. The proliferative responses of cell transfer
recipients were less than the response of TCR vaccinated donor mice,
particularly in the higher KLH doses. Some background proliferation in
the absence of KLH stimulation was observed in both cell transfer
groups. The mice were challenged with C6VL tumor 4 days after cell
transfer. The positive control group was injected three times with
TCR-KLH in QS-21. Recipient mice that received immune lymphocytes from
TCR vaccinated donors were equally protected against C6VL tumor
challenge as compared with TCR protein vaccinated group (Fig. 9). Lymphocytes from irrelevantly
vaccinated donors showed a slight degree of nonspecific protection
compared with the PBS group.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tumor immunity is often considered to be solely mediated by the cellular immune response, in particular, the activation of cytotoxic CD8+ T cells (30, 31, 32). Humoral immune response is usually thought to have minimal contribution. In fact, some have reported that a nonprotective humoral immune response has an inhibitory effect on the induction of protective T cell-dependent tumor immunity (33). In contrast, studies in a melanoma model (34), as well as in B cell lymphoma models (3, 4) suggest that humoral immunity alone is sufficient to mediate tumor immunity. Thus, the requirement and the relative contributions of cellular and humoral immunity are different depending on the tumor system being studied.
In our present study, we characterized the immune mechanisms involved
in the tumor protection of TCR-vaccinated mice, and the influence of
immunologic adjuvants in inducing a protective immune response. Several
adjuvants have been previously reported to induce protective immune
responses in other tumor model systems and were chosen for study. Both
IL-12 and QS-21 can induce strong Th1-type immune responses,
particularly in the generation of CTL (22, 23, 35, 36).
Soluble CD40L interacts with CD40, which stimulates both B and T cell
responses (37, 38, 39, 40). GM-CSF enhances Ag presentation by
APCs such as dendritic cells, and has been shown to be an effective
adjuvant in murine tumor model systems (24, 28, 41). In
our study, GM-CSF and CD40L did not stimulate a protective immune
response (Fig. 3). This may be due to differences in the tumor models
and Ags being studied. In contrast, mice vaccinated with TCR in SAF-1,
QS-21, and IL-12 were well protected against C6VL tumor (Fig. 3 and
Table II). Analysis of Abs induced by the various adjuvants showed that
humoral responses differed quantitatively as well as qualitatively,
reflected by the total Ab titers (Fig. 1) and the Ab isotype profiles
(Table I). The magnitude of anti-C6VL TCR Ab response, however, did
not correlate with tumor protection, as CD40L and GM-CSF induced
anti-TCR Abs to similar levels compared with those of IL-12.
Analysis of Ab avidity by thiocyanate elution also showed no
significant differences among the Abs induced by various adjuvants
(Fig. 2). In contrast, we found a correlation between tumor protection
and the induction if IgG2 Abs (Table I). A mixture of IgG1 and IgG2 Abs
was produced in the protected groups. In the nonprotective groups, IgG1
Abs were produced exclusively. IgG2 Abs could either participate
directly in ADCC as well as complement fixation, or simply reflect the
induction of a Th1-type cellular immune response.
Our data demonstrate that Th1-type cellular immune response is
important in mediating tumor protection in our tumor model system, and
that immune CD8+ T cells are necessary and sufficient for
conferring protection. In an adoptive cell transfer experiment, we
showed that a single transfer of 2 x 107 T
cell-enriched immune lymphocytes from TCR-vaccinated donors was
sufficient to prevent tumor growth, and the degree of protection was
equal to mice actively vaccinated with the TCR protein (Fig. 9). In
addition, the tumor protection in TCR-vaccinated mice was completely
abrogated by in vivo depletion of CD8+ T cells (Fig. 7),
despite high level of circulating anti-C6VL TCR Ab.
Although immune competent CD8+ T cells are necessary and
sufficient for tumor protection, B cells are likely important for the
induction of a protective immune response. TCR immunization of B
cell-deficient JHD mice did not stimulate a protective
immune response against C6VL (Fig. 5). The antitumor activity of B
cells may be attributed to the production of anti-C6VL TCR Abs, or
the result of processing and presentation of tumor Ags to T cells. The
antitumor effect of passive infusion of anti-TCR Abs in the
treatment of T cell tumors, including the C6VL model, has been reported
(42, 43). However, in our tumor model system, it is
unlikely that the lack of protection in vaccinated JHD mice
was due entirely to the absence of anti-C6VL TCR Abs. Transfer of
hyperimmune globulin into JHD mice prolonged tumor survival
(Fig. 6). The protection was not completely restored, however, as
hyperimmune globulin transfer into naïve C57BL/6 mice was not
sufficient in conferring full tumor protection when compared with
actively vaccinated mice (Fig. 4). Furthermore, there is no correlation
between survival and anti-C6VL TCR Ab titers, as CD8+ T
cell-depleted C57BL/6 mice that have high levels of anti-C6VL TCR
Ab were not protected against tumor. Interestingly, we were not able to
reconstitute a complete protective immune response by transferring
hyperimmune serum into JHD mice actively vaccinated with
TCR protein, and the degree of tumor protection was not improved beyond
the effect of hyperimmune serum alone (Fig. 6). The lack of tumor
protection in TCR-vaccinated JHD mice is thus likely due to
inadequate T cell response, caused by the absence of Ag processing and
presentation by B cells. Although JHD mice were reported to
be capable of generating T cell response to highly immunogenic Ags such
as viral proteins (44), it is likely that cellular immune
response to weakly immunogenic Ags such as TCR is suboptimal. Studies
have shown that B cell-deficient mice have impaired priming to protein
Ags, leading to defective T cell response (45, 46). In our
tumor model system, B cells may serve as critical APCs in the
processing and presentation of the TCR proteins. This suggests that the
native conformation of C6VL TCR must be maintained in order to induce a
protective immune response. Indeed, vaccination using a
bacterial-derive single chain TCR protein encoding for the C6VL TCR V
regions did not protect mice against tumor challenge (unpublished
observation).
Protein vaccines are thought to be processed and presented via the
exogenous protein processing pathway, leading to class II MHC Ag
presentation and the induction of strong humoral immune response. Yet
in this study we have clearly demonstrated that TCR protein vaccines
can activate CD8+ T cells that are sufficient for tumor
protection. The activation of CD8+ T cells requires class I
MHC Ag presentation, implying that tumor-derived TCR proteins in
vaccinated mice were able to cross over into the endogenous
protein-processing pathway. TCR vaccines can induce Id-specific immune
responses, thus peptides derived from the CDR3 region that encode for
unique determinants of C6VL TCR should be important in inducing a
tumor-specific immune response. Immunogenic CDR3 peptides derived from
clonotypic TCR have been identified from malignant T cells of human
patients (47, 48). However, C6VL TCR and ß proteins
do not contain any obvious peptide candidates that fit the classical
class I binding motifs in the H-2b haplotype. This was
determined by searching the TCR sequence for classical peptide anchor
residues for class I MHC (49), as well as by entering the
TCR sequence into a peptide-binding motif database (50).
The lack of dominant class I epitopes may explain the inability to
induce in vitro CTL response against C6VL tumor cells using
cytotoxicity and cytokine release assays (data not shown).
Nevertheless, the lack of peptide candidates using computer searches
does not preclude the presence of class I epitopes. Our data clearly
show that a protein TCR vaccination strategy does not limit the type of
immune response induced, as TCR proteins can be effectively processed
and presented on both class I and class II MHC molecules. It would be
of great interest to elucidate the identity of TCR peptides that are
being recognized by immune CD8+ T cells that mediate tumor
protection.
In summary, we have characterized the tumor protection mechanisms of TCR protein vaccines in the treatment of a murine T cell lymphoma model. We found that adjuvants that induced Th1-type immune responses generated tumor protection, and CD8+ T cells are necessary and sufficient for tumor protection in this model. Based on our results presented here, future experiments should aim at biasing TCR vaccines even further to induce Th1-type immune responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. C. Wong, Department of Medicine, Division of Oncology, Room M-207, Stanford University Medical Center, Stanford, CA 94305. E-mail address:
3 Abbreviations used in this paper: PI, phosphatidylinositol; SAF-1, syntex adjuvant formulation-1; KLH, keyhole limpet hemocyanin; IL-12, recombinant murine interleukin-12; CD40L, soluble trimeric murine CD40 ligand; GM-CSF, recombinant murine granulocyte/macrophage CSF; DAF, decay-accelerating factor; PLC, phospholipase C.
Received for publication August 10, 1998. Accepted for publication November 6, 1998.
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production by anti-tumor T cells. Int. Immunol. 7:1135.This article has been cited by other articles:
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S. L. Lambert, C. Y. Okada, and R. Levy TCR Vaccines against a Murine T Cell Lymphoma: A Primary Role for Antibodies of the IgG2c Class in Tumor Protection J. Immunol., January 15, 2004; 172(2): 929 - 936. [Abstract] [Full Text] [PDF]
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D. Winter, E. Fiebiger, P. Meraner, H. Auer, C. Brna, R. Strohal, F. Trautinger, R. Knobler, G. F. Fischer, G. Stingl, et al. Definition of TCR Epitopes for CTL-Mediated Attack of Cutaneous T Cell Lymphoma J. Immunol., September 1, 2003; 171(5): 2714 - 2724. [Abstract] [Full Text] [PDF]
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). The remaining groups were injected i.p. with the globulin
fraction of 3) anti-C6VL TCR hyperimmune serum (
); and 4)
anti-38C13 Id hyperimmune serum (
). One day after Ab transfer,
mice were injected i.p. with 5000 C6VL cells and monitored for
survival.
); 2) C6VL 
). Two weeks after the last vaccination, mice
were injected i.p. with 5000 C6VL cells and monitored for
survival.
