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*
Division of Surgical Oncology, Department of Surgery and
Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33101
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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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Differentiation into memory cells is in part regulated by cytokines (4, 5) and must result in some Ag-specific T cells that are resistant to apoptosis (4, 6, 7). IL-7 and IL-15 are two cytokines that have been implicated in the regulation of homeostasis and survival of CD8+ memory cells in vivo (5, 8, 9, 10, 11, 12). Comparatively less is known concerning how the cytokine environment during the initial priming of the naive T cells programs the resulting effector CTL to develop into memory cells. In most studies of adoptive immunotherapy, IL-2 has been typically used for the ex vivo expansion of donor T cells that have been previously encountered tumor. The present study, therefore, was initiated to examine to what degree the ex vivo cytokine environment used to activate naive Ag-specific CD8+ T cells subsequently favored their development into memory cells and protect against tumor activity in adoptive immunotherapy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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OT-I TCR transgenic mice (13) were maintained by breeding heterozygous OT-I TCR transgenic mice to wild-type C57BL/6J mice. The progeny were screened by PCR for the expression of the TCR transgene. All recipient mice were C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) and were used between 6 and 9 wk of age.
Biological reagents
EL-4 is a thymoma derived from the C57BL/6 mouse
(H-2b). E.G7 cells consist of EL-4 cells
transfected with OVA cDNA (14), and these were a gift from
Dr. M. Bevan (University of Washington, Seattle, WA). These cell lines
were maintained in complete medium
(CM)3 consisting of
RPMI 1640 containing 5% FCS, glutamine (30 µg/ml), penicillin (100
U/ml), streptomycin (100 µg/ml), and 2-ME (5 x
10-5 M). OVA257–264
(SIINFEKL) (13) was synthesized by Research Genetics
(Huntsville, AL). CyChrome-anti CD8
, PE-anti-V2-TCR,
FITC-V
5.1,5.2-TCR, FITC-anti-CD8 (53.6.7),
biotin-anti-Ly6C, biotin-anti-CD44 (Pgp-1),
biotin-anti-CD62L (MEL-14), and biotin-anti-CD69 were purchased
from BD PharMingen (San Diego, CA). Biotin-anti-CD25 (7D4) was
prepared in our laboratory. The PE-labeled MHC-peptide tetramer
(H-2Kb/SIINFEKL) was provided by the National
Institute of Allergy and Infectious Diseases MHC Tetramer Facility
(Atlanta, GA).
Cell culture
Unless indicated otherwise, OT-I CTL used for adoptive transfers
were generated by the culture of OT-I splenocytes (1 x
106/well) in 24-well plates containing 1 ml of CM
containing OVA257–264 (1 nM), IL-4 (175 U/ml),
and IL-2 (50 U/ml). After 3 days in culture, the cells were washed and
recultured at 0.5 x 106 cells/well in
24-well plates containing 1 ml of CM without
OVA257–264, but with the same cytokines that
were present during initial culture. After an additional 2 days in
culture, the cells were harvested and used for in vitro analyses or for
adoptive transfer. CTL assays were performed as previously described
(15) using 51Cr-labeled E.G7 and
EL-4 cells as targets. For T cell proliferation, spleen cells (2
x 105 cells/well) were cultured with the
indicated concentration of OVA257–264 in
flat-bottom 96-well culture plates for 1–3 days. Each well was pulsed
with 1 µCi of [3H]thymidine, and 5 h
later, the cells were harvested with an automated harvester. The mean
of triplicate values that varied by less than 10% was calculated. Data
are expressed as 
cpm (i.e., cpm from experimental condition -
cpm from cultures containing culture medium alone).
FACS analysis
Cells were stained with the various Ab conjugates as previously described (15). Between 50,000 and 100,000 events were collected and analyzed for each sample using a FACScan flow cytometer (BD Biosciences, San Jose, CA) and CellQuest software (BD Biosciences).
Adoptive immunotherapy
OT-I CTL were injected via the tail vein in 0.5 ml of HBSS into
normal C57BL/6J mice. Control mice for the adoptive transfers received
only HBSS. The proportion of input cells that consisted of OT-I CTL was
determined by staining for CD8, V2-TCR, and V5.1,5.2-TCR, and
this information was used to calculate the number of OT-I CTL
adoptively transferred. E.G7 or EL-4 cells (1 x
106) were injected s.c. in 0.2 ml of HBSS at the
midline of the lower abdomen 21–28 days after adoptive transfer of
OT-I CTL. The tumor cells were freshly thawed within 6 days of
inoculation. Tumor cell growth was recorded over time. Detectable tumor
was considered to be >0.5 cm2. Tumor-free
survival curves were compared by log-rank test. A p value of
<0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We recently reported that naive T cells from the OT-I TCR transgenic mice, specific for an OVA peptide, OVA257–264, in the context H-2Kb, failed to recognize and respond to the OVA-transfected E.G7 thymoma, i.e., were immunologically ignorant, after adoptive transfer into mice bearing E.G7 as a solid tumor (15). However, the adoptive transfer of OT-I effector cells, generated in short-term cultures, inhibited the growth of E.G7 in a dose-dependent manner (15). In the current study we have continued to use this model system to study the ability of in vitro-derived OT-I CTL effector cells to develop into memory cells in vivo and to then function in antitumor immunity.
Our past work indicated that the generation of primary tumor-specific
CTL in vitro was favored by culture with exogenous IL-2 and IL-4
(16). Therefore, splenocytes from naive OT-I transgenic
mice were stimulated with OVA257–264 in the
presence of exogenous IL-2 and IL-4 for 3 days and were expanded in
these exogenous cytokines for an additional 2 days. On the fifth day,
greater than 90% of the cells consisted of CD8+
cells that coexpressed V2 and V5, characteristic of the OT-I TCR
(not shown). FACS analysis revealed that these activated cells typified
effector T lymphocytes, i.e., increased size, up-regulated expression
of CD69, CD25, and CD44, and down-regulated expression of CD62L and
Ly-6C (Fig. 1
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2,
and V5 (Fig. 2A). This
analysis indicated that the number of triple-stain-positive cells in
the spleen and lymph nodes was proportional to the number of cells
administered (Fig. 3, A and
B). In each animal, the spleen, two axillary lymph nodes,
and two inguinal lymph nodes were analyzed. In sum, in animals that
received 10 x 106 CTL, the mean number of
triple-stain-positive lymphocytes recovered from a pool of these organs
was 4.4 ± (1.6) x 106 (Fig. 3B). Given that the mean number of triple-stain-positive
cells in control mice is 0.4 ± (0.2) x
106, the estimated recovery of OT-I cells in
these organs is 40% of the input cells. If one were to extrapolate
this number to the entire peripheral lymph tissue, 
68% of the OT-I
cell persisted at this time, assuming there are 25 lymph nodes in a
mouse, all containing an equal number of OT-I cells in each
node.
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). The phenotype of the OT-I cells was further
characterized for other surface markers by gating on
CD8+ and V2+ cells (Fig. 1). Unlike the in vitro-generated OT-I CTL effector cells, the in
vivo-retained cells no longer expressed elevated levels of CD69 or
CD25, but Ly6C was substantially up-regulated. In addition, CD44
remained up-regulated, whereas CD62L reverted to a higher degree of
expression. This cell surface phenotype is consistent with that
attributed to memory T cells (17, 18, 19). In three mice
tested at 70–75 days after adoptive transfer, triple-stain-positive
(Fig. 2B) or MHC-tetramer-positive (Fig. 2C) OT-I
T cells were readily detected with a cell surface phenotype identical
(not shown) to that observed at 21–28 days after adoptive transfer.
All of these features except the elevated Ly6C expression were detected
as early as 7 days after adoptive transfer (not shown). At 21–28 days
after adoptive transfer, five of five mice tested expressed a high
level of Ly6C. However, in two mice tested at earlier time points, the
high Ly6C expression was not seen, suggesting that this aspect of the
phenotype appears relatively late.
Memory CD8 T cells often show more rapid proliferation to a lower
concentration of Ag when compared with naive T cells
(18, 19, 20, 21), and they retain CTL activity
(22, 23, 24). When compared with naive T cells, the recovered
adoptively transferred "memory" OT-I T cells proliferated to an
10-fold lower concentration (Fig. 4A) of
OVA257–264 and more rapidly (Fig. 4B). Furthermore, the freshly prepared effector cells
exhibited potent and specific CTL activity to OVA-expressing targets
just before adoptive transfer (Fig. 4C). With respect to CTL
activity by the "memory" OT-I T cells, the level of cytotoxicity
was proportional to the number of OT-I CD8+ cells
that persisted in the spleen (Fig. 4D). Thus, collectively
these data demonstrate that 21 days after adoptive transfer, the OT-I
effector cells also exhibited functional properties characteristic of
memory CD8+ T cells.
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). This protection was specific to
OVA-expressing tumors, as these mice did not mount an antitumor
response when inoculated with the parental EL-4 thymoma. Furthermore,
mice (n = 2) adoptively transferred with OT-I CTL that
rejected a primary challenge with E.G7 remained tumor-free after a
second challenge with E.G7 when reinoculated 56 and 66 days after the
first inoculation (not shown). Thus, long-lasting protective
tumor-specific immunity was not prevented by the initial response by
the persistent OT-I T cells.
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Recent data suggest that generation of CTL in the presence of IL-4
enhances their survival following adoptive transfer (25).
Therefore, we examined the effect of IL-2 and IL-4 during the in vitro
culture on the long-term fate and function of OT-I CTL. In these
experiments the OT-I T cells were stimulated with a lower dose (0.1 nM)
of OVA257–264, as this was found to be necessary
for optimal cytokine expansion of these CTL (data not shown). The
proportion of cells staining for CD8, V2, and V5 did not differ
significantly between conditions and was typically 87–95% of the
cells (not shown). When compared with naive OT-I T cells (see Fig. 1),
all effector cells up-regulated CD69, CD25, and CD44, whereas the
levels of CD62L and Ly-6C were down-regulated regardless of the
exogenous cytokine(s) added during the priming cultures (Fig. 6 and data not shown). However, the
phenotype of these effector cells was not identical, as cells cultured
in only exogenous IL-4 exhibited lower levels of CD25 and CD44. The
former result likely represents the lack of endogenous IL-2 to
up-regulate CD25 (26). Intragroup variation was sometimes
noted for the level of expression of the early activation Ag CD69 and
Ly-6C, irrespective of the exogenous cytokine added to the cultures.
Furthermore, although all effector groups displayed strong CTL activity
to E.G7, effector cells generated in the presence of IL-4 or both IL-2
and IL-4 showed stronger activity than cells expanded with only IL-2
(Fig. 6B).
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). In contrast, a markedly
lower number of triple-stain-positive cells was detected in recipients
of CTL generated in IL-4. The use of a higher dose of IL-4 did not
improve the degree of persistence of the OT-I. These findings were
confirmed by staining for the donor cells with MHC-peptide tetramer
(Fig. 7). These data indicate that stimulation with IL-2 during the
generation of CTL optimally promotes the survival of these cells upon
adoptive transfer in vivo.
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). By contrast, only a small
proportion of the persistent OT-I cells generated in IL-4 expressed
high levels of CD44 and Ly6C (Fig. 8). This finding, in conjunction
with the lower yield of persistent OT-I cells (Fig. 7), indicates that
there are 4- to 6-fold fewer cells with a memory phenotype when the
naive OT-I were solely primed ex vivo with Ag and exogenous
IL-4.
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A).
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B). It is noteworthy that the IL-4-generated OT-I effector
cells developed a therapeutic effect even though these cells
inefficiently persisted and developed a memory phenotype. Thus, these
residual cells were still competent to affect E.G7 growth. Between the
treatment groups, the difference in tumor-free survival between mice
that received OT-I effector cells generated with IL-2 and recipients of
CTL generated in IL-2/IL-4 and IL-4 (50 U/ml) was not significant.
However, the greater tumor-free survival in mice that received OT-I
effector cells generated in IL-2 when compared with mice that received
CTL primed in a high dose of IL-4 (175 U/ml) was statistically
significant (p = 0.04). The greater therapeutic
efficacy of OT-I generated in IL-2 may be related to its ability to
optimally promote differentiation of the effector CTL into persistent
memory phenotypic cells, as these effector cells exhibited the weakest
CTL activity upon adoptive transfer to tumor-bearing mice (Fig. 6). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When studying cell populations thought to represent memory T cells, it is imperative that the cells under study be distinguished from chronically stimulated effector T cells that persist secondary to Ag. Even cells present several months after adoptive transfer may display characteristics more consistent with effector cells (27). Identification of CD8+ memory cells is also problematic in some models in which T cells are adoptively transferred to lymphopenic mice. In this situation, naive CD8+ T cells acquire the phenotypic and functional characteristics of memory T cells in the absence of Ag by the process of homeostatic proliferation (28, 29, 30). In our model, we have avoided potential complications due to either Ag or homeostatic proliferation by removing extraneous Ag by washing the effector cells several days before adoptive transfer to mice with a normal pool of lymphocytes.
A number of studies have documented that exogenous cytokines influence the magnitude and quality of the resulting CD8+ effector CTL generated during in vitro culture (16, 31, 32, 33, 34). More recently, we demonstrated an essential role for cytokines for the generation of effector CTL. In the absence of IL-2R and IL-4R signaling in vitro, TCR-activated CD8+ T cells failed to differentiate into CTL, in part due to lack of expression of granzyme B, and they exhibited limited proliferative capacity (3–4 cell divisions) before apoptosis by the vast majority of the cells (35). Comparatively little is known concerning the long-term consequences of these culture conditions on CTL effector cells in vivo. Our findings demonstrate a critical role for IL-2 during in vitro generation of the effector CTL for the optimal in vivo persistence of CTL with characteristics of memory T cells. Analysis of allospecific T cells in autoimmune-prone IL-2-deficient mice and an Ag-specific CD8+ cytotoxic T cell line have suggested that IL-2 favors the development of T memory cells (36, 37). Our data directly implicate signaling through the IL-2R for development of CD8 T memory cells from naive normal Ag-specific precursor T cells. In this regard, it is important to note that the sole use of exogenous IL-4 resulted in the production of optimal numbers of CTL. However, upon adoptive transfer, these effector cells did not readily persist or express a memory phenotype. These IL-4-driven effector CTL were correspondingly less effective in antitumor immunity.
Several other studies have reported findings consistent with our
results. In a model of viral infection, Aung et al. (38)
showed that the presence of IL-4 at the time of activation diminishes
the expansion of viral Ag-specific CD8+ T cells
and inhibits the development of CD8+ T cell
memory. Similarly, Villacres and Bergmann (39)
demonstrated that the absence of IL-4 at the time of activation of CTL
increases the frequency of CD8+ memory cells 8 wk
later and enhances the effector functions of these memory cells. In our
experiments, coculture of T cells with exogenous IL-2 alone or both
IL-2 and IL-4 resulted in enhanced long-term survival and
differentiation to the memory phenotype, suggesting that signal
transduction through the IL-2R is sufficient to promote memory CTL upon
adoptive transfer in vivo. This result also indicates that IL-4 does
not actively prevent engraftment of CTL that persist with a memory
phenotype. However, our experiments do not rule out that signals
through other cytokine receptors or T cell surface proteins might also
favor the engraftment and expression of the memory phenotype by
CD8+ effector cells. Furthermore, our data do not
address whether IL-2 also functions in vivo to promote memory CTL.
IL-15, whose receptor also uses IL-2R and the common
-chain for signaling, has been shown to play an important
role in the homeostasis of CD8+ memory cells
(5, 9, 10, 11) and is also a candidate to promote memory CTL
in vivo.
Although IL-2 and IL-4 redundantly function as T cell growth factors
and promote differentiation into CTL in vitro (35, 40, 41), our data suggest that IL-2 is mandatory for promoting
memory CTL. This finding was somewhat surprising because Huang et al.
(25) reported that CD8+ T cells
activated in the presence of IL-4 in vitro persisted to a greater
degree after adoptive transfer than CTL activated in IL-2. These
persistent cells expressed high levels of CD44 and produced large
amounts of IFN- following re-exposure to peptide, suggesting that
they consisted of memory cells. However, it should be emphasized that
although these investigators initially stimulated the
CD8+ T cells in either IL-2 or IL-4 for 3 days,
all cells were expanded in IL-2 for an additional 2 days before
adoptive transfer. Thus, analogous to our experimental design, these T
cells also received signaling through the IL-2R before adoptive
immunotherapy, which we believe likely contributed to promoting memory
CTL in vivo.
Our study reveals an unappreciated potential value of adoptive antitumor immunotherapy. Besides the initial antitumor response by effector CTL, in vitro expansion using IL-2 promotes the production of memory CD8+ T cells, which may favor long-term protection against tumor recurrence. Furthermore, adoptive immunotherapy with effector CTL has been shown to overcome immunological ignorance (15), a phenomenon by which naive tumor-specific T cells simply ignore tumor-associated Ag. Nevertheless, despite these beneficial features, adoptive immunotherapy with ex vivo IL-2-expanded tumor-infiltrating lymphocytes that contain CTL is often unsuccessful in cancer patients. In addition to tumor-induced immunosuppression and outgrowth of tumor Ag escape variants, growth factor deprivation is a problem after adoptive transfer, particularly when CTL have been generated and expanded over longer periods of time (1). In the model described in this study, unlike in most therapeutic protocols, large numbers of effector cells were generated over a much shorter time frame. Therefore, successful adoptive T cell therapy may be linked to rapid production of effector T cells, using the appropriate cytokines during the ex vivo culture.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas R. Malek, Department of Microbiology and Immunology, University of Miami School of Medicine, P. O. Box 016960, Miami, FL 33101. E-mail address: tmalek{at}med.miami.edu
3 Abbreviation used in this paper: CM, complete medium.
Received for publication April 23, 2001. Accepted for publication August 6, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain pairing determines the specificity of residue 262 within the Kb-restricted, ovalbumin257–264 determinant. Int. Immunol. 4:861. knockout mice. Cell. Immunol. 185:158.[Medline]
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6 mice/group.
