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*
Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland; and
Ludwig Institute for Cancer Research, Brussels Branch, Brussels, Belgium
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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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We previously established a murine tumor model in which the Ag-specific CD8+ T cell response in vivo can be longitudinally monitored in blood by flow cytometry (13, 14, 15). DBA/2 mice injected i.p. with syngeneic P815 tumor cells (H-2d) transfected with the HLA-CW3 gene (P815-CW3) showed a dramatic expansion of CD8+ T cells expressing exclusively the Vß10 segment, and phenotypically defined as CD62L-CD45RB-CD44+ activated cells (14). The specific cytotoxic activity against the immunodominant peptide CW3 170–179, presented by the H-2Kd molecule, was found exclusively in the CD8+Vß10+ population (13).
In the present study, we have used this unique model system to address the requirement for CD4+ T cell help during the development of the CW3-specific CD8+ response, with respect to different routes of immunization. In particular, the CW3-specific CD8+ T cell response was analyzed after i.p. or intradermal (i.d.)3 immunization with tumor cells, in DBA/2 mice depleted or not of CD4+ T cells. Furthermore, immunizations were conducted either with P815 cells transfected with the complete HLA-CW3 gene, or with P815 cells transfected with a minigene coding only for the CD8 epitope CW3 170–179 (P815 miniCW3). Our results show that the CW3-specific CD8+ T cell response to tumor cells injected i.d. can develop without CD4+ T cell help, whereas help is required for the i.p. route of immunization.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transfection of H-2d mouse mastocytoma P815 with the HLA-CW3 gene has already been described (16).
A 47-bp minigene coding for the CD8 epitope 170–179 of the CW3
molecule was constructed by oligonucleotide annealing and cloned at the
unique EcoRI site of expression vector pCD-SR
(17). A
modification of the calcium phosphate DNA precipitation method of
Graham and Van der Eb (18, 19) was used to transfect P815-HTR cells
with the CW3 minigene cloned into pCD-SR. Briefly, a calcium
phosphate DNA precipitate, containing cDNA clone pCD-SR-CW3 minigene
and selective plasmid pSVtk-neoß (20) at a ratio of 10:1, was
allowed to form for 30 to 45 min at room temperature. A quantity
amounting to 5.106 P815 cells was centrifuged for 10 min at
300 x g, and the cell pellet was resuspended directly
in the calcium phosphate DNA precipitate containing 20 to 25 µg total
DNA in 2.5 ml. The mixture was incubated for 30 min at 37°C and then
added to an 80-cm2 flask (Nunc, Roskilde, Denmark)
containing 22.5 ml DMEM (Life Technologies, Paisley, U.K.) supplemented
with 10% heat-inactivated FCS (Irvine Scientific, Santa Ana, CA).
After 24-h incubation at 37°C, cells were centrifuged and resuspended
in 40 ml fresh medium. Forty-eight hours after transfection, cells were
washed and selection was conducted in DMEM supplemented with 10%
heat-inactivated FCS and 1.5 mg/ml G418 (Geneticin; Life Technologies).
Transfected cells were subsequently cloned at 1 cell/well in the
selection medium, and clones were screened in a cytolytic assay with
the CTL clone 1.1 specific for the CW3 peptide 170–179 presented by
H-2Kd (21).
Mice and immunizations
Adult female DBA/2 mice (Harlan Olac, Bicester, U.K.) were injected i.p. or i.d. on the back with, respectively, 107 or 2.107 viable P815 tumor cells transfected with the complete HLA-CW3 gene (P815-CW3) or the CW3 minigene (P815 miniCW3). Both tumor cells were maintained as an ascitic tumor passaged weekly in nude mice. For some experiments, mice were boosted with 107 P815-CW3 cells injected i.p. At various times after primary immunization or challenge, mice were bled by the tail vein and PBL were isolated by Ficoll-Hypaque gradient centrifugation (Pharmacia Biotech, Uppsala, Sweden).
In vivo CD4 depletion
Mice were injected i.p. with anti-CD4 mAb (GK1.5 ascites) diluted at 1/20 in 0.2 ml PBS, on days -3, -2, -1, +3, +7, and +11, and immunized at day 0. Residual CD4+ T cells represented less than 1 to 2% of total T cells at the time of immunization, and this level remained constant throughout the primary response. Control mice were injected with PBS on the same schedule.
Flow cytometry analysis
Triple stainings were performed on PBL with FITC-conjugated anti-CD62L (Mel-14, prepared in our laboratory), phycoerythrin-conjugated anti-CD8 (53.6.7; Boehringer Mannheim, Mannheim, Germay), and biotinylated anti-Vß10 (B21.5, prepared in our laboratory) revealed with streptavidin-Tricolor (Caltag Laboratories, San Francisco, CA). Samples were analyzed on a FACScan equipped with LYSIS II software (Becton Dickinson, San Jose, CA).
For cell-sorting experiments, PEL and spleen cells were triple stained, as indicated above, and CD8+CD62L-Vß10+ or Vß10- cells were sorted on a FACStar (Becton Dickinson), collected in DMEM supplemented with 5% heat-inactivated FCS, and directly tested for ex vivo cytolytic activity.
Cytolytic assay
Mice were killed 2 wk after i.p. immunization with P815-CW3 cells. PEL were purified by nylon wool columns, and single cell suspensions of splenocytes were prepared by standard procedures. Activated CD8+ T cells were sorted on the basis of Vß10 expression, as described above, and used as effector cells in the cytolytic assay.
P815-CW3 and control P815 cells were labeled with 100 µCi of 51Cr, as previously described (22), for 1 h at 37°C, and washed five times. A quantity amounting to 2.103 51Cr-labeled target cells was mixed with varying numbers of freshly sorted effector cells in V-bottom microplates, in DMEM supplemented with 5% heat-inactivated FCS. 51Cr release in supernatant was measured after 4-h incubation at 37°C. The percent specific lysis was calculated as described (22).
In some experiments, CTL clone 1.1 (21), specific for the CD8 epitope 170–179 of HLA-CW3, was used as effector cell.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous reports from our laboratory have shown that the expanded
CD8+Vß10+ T lymphocyte population in
HLA-CW3 immune DBA/2 mice showed significant ex vivo CW3-specific
cytolytic activity, whereas CD8+Vß10- cells
did not (13). To rule out the presence of CW3-specific CTL expressing
another Vß segment than Vß10 in the activated
CD8+CD62L- population,
CD8+CD62L-PEL or splenocytes (isolated from
DBA/2 mice immunized i.p. with P815-CW3 cells) were sorted on the basis
of Vß10 expression and subsequently tested for CW3-specific cytolytic
activity. As shown in Figure 1
, both in
PEL and spleen, CW3-specific cytolytic activity was only detected in
the CD8+CD62L-Vß10+ population,
even at high E:T ratio. Thus, the phenotypically defined
CD8+CD62L-Vß10+ T cell
population will hereafter be referred to as CW3-specific CTL.
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Intradermal immunization with P815-CW3 tumor cells led to a very
strong CW3-specific CTL response in blood, similar to what was observed
after i.p. immunization (Table I, Fig. 2): Vß10+ cells
represented 70% on average, and up to 80% in some experiments, of the
activated CD8+CD62L- T cells in PBL, as
compared with 7% in naive mice. The percentage of CD62L-
cells in the CD8+ population also notably increased,
whereas the proportion of CD8+ T cells only increased
marginally as compared with control mice (Table I).
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). Differential expansion of CW3-specific CTL following i.p. or i.d. immunization with P815 cells transfected with a CW3 minigene
To address a potential role for a CD4 helper epitope in the
development of the CW3-specific CD8+ T cell response,
we used P815 tumor cells transfected either with the complete
HLA-CW3 gene (P815-CW3 cells), or with a minigene coding
only for the CD8 epitope 170–179 (P815 miniCW3 cells). As expected,
both tumor cells were efficiently recognized and lysed by a CTL clone
specific for the CW3 peptide 170–179 presented by H-2Kd
(Fig. 3), but only the P815-CW3 cells
expressed HLA-CW3 on the surface.
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), although it was significantly
different as compared with naive mice (Student’s t test,
p < 10-4). In marked contrast, i.d.
immunization with P815 miniCW3 cells led to a strong CW3-specific
response, as on average, more than 40% of the
CD8+CD62L- population expressed Vß10 (Fig. 2). These data suggest that development of the CW3-specific CD8
response is not strictly dependent on a potential CD4 epitope in the
HLA-CW3 molecule when tumor cells are injected i.d. Kinetics of the CW3-specific CD8 response after i.p. or i.d. immunization
Although i.d. immunization with P815 miniCW3 cells led to a very
strong expansion of CW3-specific T cells, the response remained lower
than with P815-CW3 cells injected i.p. or i.d. Kinetics experiments
confirmed and extended these observations. As shown in Figure 4, i.p. or i.d. immunization with
P815-CW3 cells was followed by a very rapid expansion of CW3-specific T
cells, as the percentage of Vß10+ cells reached 50%
of the CD8+CD62L- population on day 8 after
immunization, and it was maximum on day 12. Furthermore, with both
routes of immunization, the level of CW3-specific T cells remained
extremely high during a long period of time, with only a slight
decrease 45 days after immunization. As compared with the response
induced by P815-CW3 cells, i.d. immunization with P815 miniCW3 cells
also led to a strong expansion of the CW3-specific population, but the
increase was slower, the peak of the response was slightly delayed, and
the percentage of CW3-specific T cells decreased more rapidly after the
peak of the response.
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As shown above (Figs. 2 and 4), immunization of DBA/2 mice
with P815 cells transfected with the CD8 epitope alone of the HLA-CW3
molecule (P815 miniCW3) led to a strong CW3-specific T cell response
only when the tumor cells were injected i.d. Since the P815 miniCW3
cells cannot express an HLA-CW3-encoded helper epitope, these data
suggest that the CW3-specific CD8 response elicited by i.p. and i.d.
routes of immunization differs in the requirement for CD4 T cell help.
As an independent test of this hypothesis, mice were depleted in vivo
of CD4+ T cells by injections of anti-CD4 mAb
before immunization with P815-CW3 or P815 miniCW3 cells. The CD4
depletion was essentially complete at the time of immunization
(<1–2% of residual CD4+ T cells) and remained maximum
throughout the primary response (data not shown).
As shown in Figure 5A, CD4
depletion abrogated the CW3-specific T cell response against P815-CW3
injected i.p. In contrast, the CW3-specific CD8 response after i.d.
immunization with P815-CW3 or P815 miniCW3 was similar in the
CD4-depleted mice as compared with the nondepleted controls (Fig. 5A). Indeed, statistical analysis of the percentage
of Vß10+ cells in the CD8+CD62L-
population after i.d. immunization showed no significant difference
between mice depleted or not of CD4+ T cells (Student’s
t test, p > 0.05), whereas the difference
was highly significant after i.p. immunization (Student’s t
test, p < 10-6). Surprisingly, expansion
of CW3-specific CD8+ T cells was increased significantly
after CD4 depletion in mice injected i.d. with P815 miniCW3 cells
(Student’s t test, p = 10-4),
reaching the same level observed in mice injected with P815-CW3 cells
(Fig. 5B).
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). In contrast, survival of mice
injected i.d. with both types of tumor cells was not affected by CD4
depletion (Fig. 6 and data not shown). The clear correlation between
the percentage of Vß10+ cells in the
CD8+CD62L- population and the ability to
reject tumor cells confirms and strengthens the finding that
CW3-specific CTL exclusively express the Vß10 segment (Fig. 1)
(13).
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As mentioned above, the murine model used in this study
allows us to monitor the specific CD8 response ex vivo by flow
cytometry. We could thus analyze longitudinally in the same individual
mice the CW3-specific CD8 primary response and the memory response to a
secondary challenge, to determine whether the absence of CD4 help
during the primary CD8 response would modify the establishment of CD8
memory. To standardize the readout, CD8 memory was always measured as
the early response (6 days after injection of tumor cells) to an i.p.
challenge with P815-CW3 cells. Groups tested included mice immunized 3
mo before with P815-CW3 or P815 miniCW3 injected i.p. or i.d., and
depleted or not of CD4+ T cells throughout the primary
response. Nonimmunized mice used as controls for the primary response
were divided into two groups. One group was retained as naive controls,
while the other group was injected i.p. with P815-CW3 cells at the same
time and in the same conditions as the immune mice. As expected, no
CW3-specific response could be detected in control mice 6 days after
the primary immunization, whereas a strong CW3-specific expansion was
observed after 14 days (Fig. 7,
inset).
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). The strongest CW3-specific memory response was
observed in mice immunized with P815-CW3 cells during the primary
response. This response was similar between groups of mice immunized
i.p. or i.d., and CD4 depletion during the primary response (after i.d.
immunization) did not alter the magnitude of the memory response
(Fig. 7). Furthermore, the percentage of CW3-specific memory T cells
observed 6 days after challenge in these groups of mice was comparable
with the CW3-specific response in control mice 14 days after primary
i.p. immunization with P815-CW3 cells. Mice immunized during the
primary response with P815 miniCW3 i.p. or i.d. also displayed an
efficient CW3-specific CD8 memory response, although it was lower than
in mice immunized with P815-CW3 cells (Fig. 7). Finally (similarly to
what was observed during the primary response (Fig. 5B)), CD4 depletion during the primary response to
P815 miniCW3 cells injected i.d. led to a higher CD8 memory response as
compared with nondepleted mice. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The precise nature of the putative HLA-CW3-specific helper epitope recognized by CD4+ T cells on P815-CW3 remains to be determined. In this regard, i.p. injection of P815 miniCW3 cells cotransfected with the intact HLA-B7 gene did not restore the CW3-specific CD8+ T cell response in DBA/2 mice (data not shown), suggesting that the putative helper epitope is not shared between HLA-CW3 and HLA-B7. Additional experiments using synthetic peptides derived from nonhomologous regions of these two HLA molecules will be required to formally demonstrate the existence of a CW3-specific helper epitope.
In our model system, the development of CW3-specific CD8 memory also does not appear to require CD4+ T cell help during the primary response, i.e., during the activation and expansion phase of CW3-specific naive precursors. Signals necessary to rescue CW3-specific effector CD8+ T cells from activation-induced cell death can thus be provided in the absence of CD4 help. This result further strengthens the hypothesis that CD4+ T cell help in the CW3 system is not mediated by a direct effect on CW3-specific CD8+ T cells. In addition, the number of secondary effector CD8+ T cells decreases much more rapidly after the peak of the response as compared with primary effectors. This phenomenon could reflect a reduced tumor load in the secondary response. Indeed, the CD8+ response after boosting is much faster than the primary response, so that tumor cells could undergo a smaller number of divisions before their clearance. Alternatively, this result could reflect a differential susceptibility to activation-induced cell death of primary effectors derived from naive CD8+ T cells and secondary effectors derived from the memory pool.
Whereas in vivo depletion experiments showed that CD4 help was not required for the i.d. route of immunization with P815-CW3 cells, similar immunization with P815 miniCW3 cells revealed an increased expansion of specific CD8+ T cells after CD4 depletion. To explain this surprising result, we propose the following hypothesis: both an HLA-CW3-encoded helper epitope and a regulatory epitope encoded by the P815 cell itself, respectively recognized by helper and regulatory CD4+ T cells, could coexist in the P815-CW3 cells. The very effective CD8 response following i.p. immunization, in which CD4 help is necessary, further suggests that the HLA-CW3 helper epitope would be dominant over the putative P815 regulatory epitope. In P815 miniCW3 cells, only the P815 regulatory epitope remains, which would explain the increased CW3-specific CD8+ response after CD4+ T cell depletion in the i.d. route of immunization, insofar as CD4 help is not required. Our hypothesis is also supported by previous reports of CD4+ suppressor T cells specific for the P815 tumor cells (30, 31), as well as evidence of regulatory CD4+ T cells in several diseases including chronic experimental autoimmune encephalomyelitis (32), inflammatory bowel disease (33, 34), and contact sensitivity (35).
Our finding that an efficient Ag-specific CD8+ T cell response can develop without CD4 help after i.d. immunization might have important implications for vaccination strategies aiming at stimulating antitumor immunity in humans, since the skin represents a very convenient injection site for clinical purposes. In this regard, it is of interest that Sampson et al. (36) showed that s.c. vaccination with irradiated granulocyte-macrophage CSF-producing tumor cells increased survival of mice bearing preestablished tumors in the brain, conceivably by stimulating uptake and presentation of tumor Ags by cutaneous dendritic cells. The tumor cells used in this particular study were poorly immunogenic, and the beneficial effect observed with granulocyte-macrophage CSF-producing tumor cells was not dependent on CD4+ T cells, thus suggesting that our results obtained with the immunogenic tumor P815-CW3 might be extended to other less immunogenic tumors. Several other studies based on expression of costimulation molecules such as B7-1 or B7-2 and ICAM-1 (37, 38, 39), or administration of cytokines such as IL-12 (40), which all play a fundamental role in dendritic cell function, demonstrated rejection or regression of established weakly immunogenic tumors independently of CD4+ T cells. In the same context, s.c. injection of dendritic cells transduced with a model tumor Ag gene was highly efficient in treatment of established tumors expressing this Ag (41). Together with our results, these studies suggest a key role for cutaneous dendritic cells (involved in uptake and presentation of tumor Ags) in the development of an efficient helper cell-independent CTL response against tumors.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. H. Robson MacDonald, Ludwig Institute for Cancer Research, Lausanne Branch, 155, Chemin des Boveresses, CH-1066, Epalinges, Switzerland.
3 Abbreviations used in this paper: i.d., intradermal; CD62L, CD62 ligand; PEL, peritoneal exudate lymphocyte.
Received for publication October 28, 1997. Accepted for publication February 5, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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