Low avidity antigen-specific CTL clones have poor in vitro and in vivo anti-tumor effects
Low avidity effector T cells can recognize peptide-pulsed target cells but often fail to recognize endogenous antigens on tumor cells (12
). We therefore analyzed the in vitro
and in vivo
activity of a high and low avidity CTL clones (clone 24 and clone 8) which are both specific for the cognate antigen, TRP-2180–188
presented by H-2Kb
. As expected clone 8 produced significantly less IFN-γ than clone 24 when stimulated with B16 tumor cells () However when B16 tumor cells, transfected with the costimulatory molecule B7.1 (B16-B7.1), were used as targets, clone 8 produced large amounts of IFN-γ ().
In vitro and in vivo activity of high and low avidity TRP-2-specific CTL clones
We also found marked differences in vivo between clone 24 and clone 8. When analyzed in the B16 lung metastasis model (see M&M), only the high avidity CTL, clone 24, efficiently treated lung metastases while no significant difference was found between clone 8 treated and untreated mice (). Therefore, clone 8 represents a classic low avidity anti-tumor CTL which is inefficient at eradicating tumors.
aAPC reverse in vivo inefficacy of the low avidity anti-tumor CTLs
To study the effect of aAPC, we tested the ability of aAPC to augment the efficacy of adoptively transferred, low avidity CTL clone 8. For these studies we generated aAPC in which signal 1, peptide-loaded Kb
-Ig complexes, and signal 2, B7.1-Ig complexes, are covalently coupled to beads, see Suppl. Fig. 1
for schematic and details of all aAPC used for these studies. In mice previously injected with B16 tumor, we examined whether in vivo
aAPC-activated clone 8 could eradicate lung metastases. As schematically shown in , on day 3 after B16 tumor cell injection, B6 mice received 5×106
TRP-2-specific CTLs and were injected i.v. on day 4, 5, and 6 with 107
aAPC/mouse and all mice were sacrificed 14 days later. Administration of cognate TRP-2 peptide-loaded aAPC led to complete eradication of the tumor by clone 8 which was otherwise ineffective ().
Cognate TRP-2 aAPC treatment enhances in vivo anti-tumor activity of low avidity CTL clones
To analyze the relevance of signal 1 and signal 2 on the aAPC, we tested the effect of administration of the following aAPC; 1) signal 1 aAPC, 2) signal 2 aAPC, and 3) non-cognate SIY aAPC (see Suppl. Figures 1
). No anti-tumor effect was seen in mice treated with the signal 1 aAPC, signal 2 aAPC, or non-cognate SIY aAPC demonstrating that both cognate signal 1 and signal 2 were required for effective in vivo
activation of low avidity CTLs (). Additionally, no anti-tumor effect was seen in mice that were immunized with cognate TRP-2 aAPC only. These results indicate that aAPC provide more than basic costimulation to increase the efficiency of tumor immunotherapy.
To analyze the requirements for antigen-specific activation, a β-galactosidase-specific (β-gal) CTL clone was adoptively transferred to mice () and mice were injected with either β-gal or SIY aAPC (). No effect was observed on metastasis formation, demonstrating that non-specific CTLs exert no anti-tumor activity.
aAPC induce in vivo proliferation of adoptively transferred TRP-2 specific CTLs
To further analyze the effect of aAPC, we evaluated in vivo proliferation of the low avidity CTLs. Tumor-bearing or tumor-free CD45.1+ B6 mice were adoptively transferred with 5×106 CFSE-labeled, CD45.2+ clone 8 cells. After 3 days, mice were immunized with either cognate TRP-2 aAPC or non-cognate SIY aAPC. Seven days later all mice were sacrificed and organs were collected and analyzed for the presence and expansion of CD45.2+/CD8+ cells. Transferred CTLs were found only in the lung, which could be related not only to the route of administration but, also, to the specific homing of the CTLs to the tumor site.
Administration of cognate TRP-2 aAPC induced significantly more proliferation of the transferred clone 8, than the non-cognate SIY aAPC in tumor-bearing mice as determined by CTL expansion () and CFSE dilution (). Interestingly, low avidity, TRP-2-specific clone 8 underwent more cell divisions in the presence of cognate TRP-2 aAPC in tumor-bearing mice (). The majority (~80%) underwent 5 or more cell divisions and only 2.2% of the transferred CTLs did not proliferate. In contrast, significantly less expansion was observed in mice treated with non-cognate SIY aAPC (35.4% of the transferred CTLs remained undivided and only ~20% underwent more than 3 cell divisions). These data demonstrate that aAPC can be used to efficiently expand adoptively transferred CTLs in tumor-bearing mice. The reduced CTL proliferation in mice treated with the non-cognate SIY aAPC () further confirms the finding that the B7.1 alone is not sufficient to induce anti-tumor activity or proliferation of the low avidity CTL clone.
In vivo proliferation of TRP-2-specific CTL clones after antigen-specific aAPC injection
Interestingly, TRP-2-specific CTL expansion triggered by cognate TRP-2 aAPC in the lung was more vigorous in tumor bearing mice, while only a limited expansion was observed in tumor-free mice (, left panels). This effect might be related to the biodistribution of aAPC. Alternatively, it is also possible that, in the absence of tumor, activated T cells did not localize to the tumor site but distributed throughout the animal and were too dilute to be detected. To investigate whether the differences in proliferation between tumor-free and tumor-bearing mice were dependent on differences in the in vivo distribution of the aAPC, we injected tumor-bearing and tumor-free mice with aAPC and analyzed tissue distribution of aAPC in various organs. On day 7, we found ~40% fewer aAPC in the lungs of tumor-free mice than in the lungs of tumor-bearing mice and the absolute number of aAPC was further reduced by day 14 (). In contrast, no differences were observed in the number of aAPC in other organs. Therefore, the presence of the tumor affects the biodistribution and persistence of aAPC, which may explain the increased T cell proliferation in lungs of tumor-bearing mice.
TRP-2-specific aAPC enhance in vitro activity of low avidity CTLs
To further analyze the effect of aAPC, we performed in vitro assays including effector cytokine secretion and TCR down-regulation assays on the TRP2-specifc clones. Both low and high avidity clones secreted IFN-γ at comparable levels after cognate TRP-2 aAPC stimulation (). The amount of IFN-γ detected for non-cognate SIY aAPC, signal 1 and signal 2 aAPC was comparable to non-stimulated CTL clones (). Only cognate TRP-2 aAPC induced significant effector functions in low avidity CTLs, clone 8. Analogously, anti-βgal CTLs were only stimulated to release IFN-γ by either βgal96–103 peptide-pulsed targets or βgal-aAPC, whereas there was no recognition even of B16-B7.1 melanoma targets ().
Cognate TRP-2 aAPC enhance in vitro activity of low avidity CTL clones
T cell activation was also evaluated by TCR down-regulation after stimulation with either, peptide-pulsed cells, plate bound anti-CD3 mAb, or aAPC. TCR down-regulation was more sensitive in the high avidity CTLs, clone 24, than in the low avidity CTLs, clone 8, when stimulated with peptide-pulsed target cells (). The limited TCR down-regulation for clone 8 CTLs was not due to an overall impairment of the signaling machinery since stimulation with plate bound anti-CD3 induced strong TCR down-regulation in both clone 8 and clone 24 CTLs (). Cognate TRP-2 aAPC efficiently induced similar TCR down-regulation in both CTL clones while signal 1 aAPC did not induce any TCR down-regulation (). In summary, cognate TRP-2 aAPC are effective stimulators of activation of low avidity CTLs.
We determined whether low avidity CTLs stimulated with cognate aAPC in vitro might regain the capacity to recognize B16 tumors not expressing B7.1. Clone 8 and 24 CTL were tested immediately (Day 0, ) or 4 days following in vitro stimulation with cognate aAPC at 1:1 ratio (Day 4, ). Although on day 4 we detected a slightly higher reactivity in terms of IFN-γ released in culture upon stimulation, the functional response of clones 8 and 24 was not affected, i.e. clone 24 recognized efficiently both B16 melanoma and the B7.1 transfected variants whereas clone 8 only responded to B16-B7.1 cell stimulation (). As expected, there was also no change in the overall CTL functional avidity as measured by peptide titrations (data not shown).
aAPC injection increases in vivo anti-tumor responses of low avidity, telomerase-specific CTLs
We analyzed the efficacy of aAPC in another clinically relevant system, a polyclonal mouse CTL line specific for mouse telomerase (TERT-CTL)(24
). TERT-CTLs were greater than 95% CD8+ but only 36% antigen-specific, as determined by tetramer staining (data not shown).
TERT-CTLs also did not efficiently recognize B16 melanoma cells in vitro, but they did recognize peptide-pulsed target cells and B16-B7.1 targets (). Incubation of TERT-CTLs with cognate m-TERT aAPC but not control aAPC induced significant release of IFN-γ, similar to the low avidity TRP-2-specific clone.
aAPC enhance the in vitro and in vivo activity of m-TERT specific CTLs
To evaluate the aAPC-based in vivo stimulation of the adoptively transferred TERT-CTLs, we administered TERT peptide-loaded aAPC (see schematic). While adoptive transfer of TERT-CTLs alone induced some tumor reduction (), injection with cognate TERT-aAPC led to a significant decrease in tumor burden (p=0.0001). As expected, non-cognate aAPC did not reduce the tumor burden nor did TERT-aAPC alone, without adoptively transferred CTLs (). Thus treatment with cognate TERT-specific aAPC also led to significant reduction in tumor burden in vivo.
One potential reason for incomplete tumor clearance could be epitope loss due to immunoselection by the adoptively transferred CTL. To examine this possibility, we isolated tumor after treatment with mTERT-specific CTL and aAPC and analyzed the ability of the mTERT-specific CTLs to recognize the tumor isolated from the various mice. There was no difference in recognition, by TERT-specific CTLs, of the tumors isolated from animals injected with cognate TERT-specific aAPC or non-cognate aAPC (Supplementary Figure 3
). Thus alternative explanations including dosing of aAPC, activity of polyclonal TERT antigen-specific CTLs and level of TERT antigen on tumor cells may account for residual tumors in the TERT-system.
Mechanistically, we tested if aAPC directly stimulate low avidity CTL activity. For these studies, we used the low avidity mTERT-specific CTL and clone 8. CTL were incubated with 51
Cr labeled B16 tumor cells, either alone or in the presence of cognate aAPC or non-cognate aAPC. Significant increases in CTL-mediated killing were seen in cognate aAPC-treated CTLs as compared to non-cognate SIY-aAPC (). Similar results were obtained for clone 8 while no effect was seen on the high avidity clone 24 response (Supplemental Figure 4
). Thus aAPC stimulation leads to enhanced killing by low avidity CTL toward tumors that otherwise would not be efficiently recognized.
aAPC administration reduces tumor growth in a subcutaneous tumor treatment model
To evaluate the impact of aAPC in a treatment model of subcutaneous tumors, we injected B16 tumor cells s.c. to induce solid tumors. Once tumors reached a size of ~10 mm2, hgp10025–33-specific CTLs were injected and mice were treated with either control or cognate aAPC. Administration of cognate aAPC led to a significant reduction in tumor growth (p=0.038) as compared to the control groups that were treated with either non-cognate aAPC or IL-2 alone (). Kaplan-Meier survival analysis () also revealed statistically significant differences between cognate aAPC treatment and non-cognate aAPC (P=0.0046) and between cognate aAPC and animals receiving just CTLs alone and IL2 (P<0.001). In contrast there was no significant influence of treatment with non-cognate aAPC compared to untreated animals. Thus aAPC treatment is also effective in reducing growth of an established subcutaneous tumor.
Therapeutic activity of aAPC against subcutaneous tumors