CD8+ T cells optimally suited for anti-tumor ACT should be able to travel to the site of the tumor, recognize the malignant cells, and erradicate them. In addition, some of the transferred CTL should persist as memory CTL lifelong to ensure anti-tumor immunity. Here, we described a simple strategy for in vitro CTL priming and host conditioning that fulfills these demands based on its reproducible tumor eradication and generation of immunity in a melanoma mouse model known to be fairly resistant to immunotherapy.
Our study investigated in detail the role of the specific stage of CTL activation/differentiation at the time of ACT and their transition into long-term anti-tumor immunity in vivo.
It is worthwhile mentioning that our differentiation strategy employed naïve CTL, rather than tumor infiltrating lymphocytes (TIL). The former, but not the latter, are easily obtained and may provide more reproducible results because TIL of late stage tumors may be dysregulated by multiple mechanisms [47
]. On the other hand, the choice of naïve T-cells requires redirecting their specificity to known tumor antigens. Experiments addressing the latter strategy are underway in several laboratories, including ours.
Our investigations demonstrated that an early stage of CTL activation (day 3) can be differentially programmed by cytokine to benefit from the environment of CTX treated hosts. Only cells primed in the presence of IL-12, but not IL-2 ( and data not shown), achieved the therapy goal. Likewise, extending the in vitro culture to a total of seven days generated a different cell stage (MD-M, MP and MGL; unpublished observation) and failed to eradicate the tumor ().
Over the past decade many elegant studies have shed molecular light on how activation of CTL proceeds in vivo
upon viral infection. An early stage has been defined as memory precursors that are IL-7R high and KLRG1 low [43
]. During further activation and differentiation in vivo
most of them may acquire cytotoxic activity and eventually become terminal effectors before they die or exhaust [46
] while few will survive to give rise to long-term memory CTL. Unexpectedly, we found that the cells most effective in the adoptive transfer acquired an unusual gene expression profile if they represented an identical stage at the time of their analysis by real-time RT-PCR rather than a combination of distinct populations. The former, but not the latter, is supported by the results from the flow cytometry because the histograms primarily displayed single populations ( and data not shown). Transcription factor expression, such as prdm1, and surface molecule expression such as CD25 (), corresponded to strongly activated effector CTL while klrg1 and p19arf accumulation suggested they were terminally differentiating into senescent and/or exhausted cells [46
]. Similarly perplexing, they expressed CD62L suggesting that they are homing to lymph nodes thought to be reserved for quiescent naïve and central memory CTL. Interestingly, the high expression of t-bet by these cells has been associated previously not only with the effector stages of T-cells but specifically also to their homing to tumors in situ
]. Taken all together, our in vitro
activation of CTL seems unlikely to proceed analogously to their physiological activation in vivo.
Perhaps it should be expected that CTL capable of efficiently seeding CTX treated hosts are distinct from CTL stages defined according to anti-viral CTL responses. Additional correlative experiments in our laboratories point to a unique role of the tumor suppressor p19arf for the IL-12 dependent in vitro
activation/ differentiation (M.D.-M., M.P. and M.G.L. unpublished observation).
The success of the ACT strategy was contingent on a synergy between the use of IL-12 to prime naïve CTL and the conditioning of the host with CTX. Notably, a lymphodepletion regimen of a single bolus of CTX without a second drug or total body irradiation was sufficient to convert the limited anti-tumor activity of transferred early Pmel12
into their capability of complete tumor rejection and, ultimately, anti-tumor immunity. This finding could have important clinical implications because it is often suggested that the efficacy of adoptively transferred T cells is proportional to the intensity of lymphodepletion [52
] while intensification comes at the expense of higher toxicities. We are currently addressing whether the IL-12 primed CD8+
T cells are “supraoptimally fit” and thus require a less rigorous conditioning regimen. Our current working hypothesis is that the synergistic action is due to the in vitro
generation of a transient CTL stage that is optimally responsive in vivo
to the cytokine milieu in the CTX treated host [54
]. Based on the known pleiotropic effects of CTX, other explanations may include its induction of lymphopenia [55
], its reduction of Tregs [56
] and its systematic activation of DC or even its induction of a highly immunogenic form of tumor cell lysis [57
]. These explanations are not exclusive of each other and individual mechanism may become critical at different time points after the adoptive transfer.
Our results show a strong correlation between the levels of circulating donor cells and tumor regression with ineffective populations disappearing by day 8 after the ACT (). Similar findings have been described in patients receiving ACT for melanoma. In these patients, transferred cells with long-term persistence in circulation expressed high levels of CD127 (IL-7R), which suggested an IL-7-dependent survival mechanism [58
]. The cells we used for ACT did not significantly upregulate the IL-7R () and it was upregulated only among their long-term surviving progeny (). Similarly, the transcription factor TCF-7 that is known to promote long-term memory and longevity of memory CTL [38
], was down regulated in the donor cells, but it became the most highly induced gene in their long-term surviving progeny . The high expression of TCF-7 and IL-7R by the Pmel cells remaining in the animals for at least nine months after cure (data not shown) led us to investigate whether long-term survival of the adoptively transferred CTL became associated with the acquisition of memory stem-cell like traits that were reported to reside within a CD44low
]. Indeed, we found that a significantly larger population of CD44low
cells existed among the donor population compared to the endogenous CTL (). These cells also preferentially homed to the lymph node and bone marrow (), the two organs in which memory CTL are preferentially maintained [28
]. Functionally these cells were even able to efflux daunorubicin and to propagate themselves upon lymphopenia-inducing cytotoxic chemotherapy, a trait they share with hematopoietic stem cells [59
]. This cumulative evidence suggests that the donor derived CTL evolving after ACT had acquired stem-cell like features necessary to maintain long-term memory in vivo.
Finally, these cells were readily responsive to a second antigen and tumor challenges ( and ).
In conclusion, this report describes a simple and extremely potent strategy for ACT of CD8+ T -cells in a pre-clinical mouse melanoma model. The two key components entail the brief activation of naïve CTL in the presence of IL-12 in vitro and their transfer into tumor-bearing animals treated with a single dose of CTX. The key results include that an apparently novel differentiation stage of the transferred CTL promotes both tumor eradication and the generation of self-renewing memory CTL. Our results also suggest that transcription factors driving CTL activation and differentiation may become useful bio-markers to predict the efficacy of cells for ACT prior to their infusion and that a threshold of donor cells needs to be detectable in the blood circulation within the first week long term tumor eradication.