Background Clinical application of adoptive T cell therapy (ACT) has been hindered by an inability to generate adequate numbers of non-tolerized, functionally active tumor-specific T cells which can persist in vivo. In order to address this, we evaluated the impact of IL-12 signaling during tumor-specific CD8+ T cell priming in terms of persistence and anti-tumor efficacy using an established B16 melanoma tumor adoptive therapy model. Study Design B6 mice were injected subcutaneously with B16 melanoma tumor cells. On day 12 of tumor growth, mice were preconditioned with cyclophosphamide (4mg dose, i.p.), and one day later, treated by adoptive transfer of tumor-specific pmel-1 CD8+ T cells primed ex vivo 3 days earlier with (i) both IL-12 and antigen (hGP10025–33 peptide) or (ii) antigen only. Tumors were measured biweekly and infused donor T cells were analyzed for persistence, localization to the tumor, phenotype, and effector function. Results Adoptive transfer of tumor-specific CD8+ T cells primed with IL-12 was significantly more effective in reducing tumor burden in mice preconditioned with cyclophosphamide compared with transfer of T cells primed without IL-12. This enhanced anti-tumor response was associated with increased frequencies of infused T cells in the periphery and tumor as well as elevated expression of effector molecules including granzyme B and interferon-γ (IFNγ). Conclusions Our findings demonstrate that ex vivo priming of tumor-specific CD8+ T cells with IL-12 dramatically improves their in vivo persistence and therapeutic ability upon transfer to tumor-bearing mice. These findings can be directly applied as novel clinical trial strategies.

IL-2 is a lymphocyte growth factor that is an important component of many immune-based cancer therapies. The efficacy of IL-2 is thought to be limited by the expansion of T regulatory cells, which express the high affinity IL-2 receptor subunit, IL-2Rα. IL-15 is under investigation as an alternative to IL-2. Although both cytokines signal through IL-2Rβγ, IL-15 does not bind IL-2Rα and therefore induces less T regulatory cell expansion. However, we found that transferred effector CD8+ T cells induced curative responses in lymphoreplete mice only with IL-2-based therapy. While conventional in vitro assays showed similar effector T cell responsiveness to IL-2 and IL-15, upon removal of free cytokine, IL-2 mediated sustained signaling dependent on IL-2Rα. Mechanistically, IL-2Rα sustained signaling by promoting a cell-surface IL-2 reservoir and recycling of IL-2 back to the cell surface. Our results demonstrate that IL-2Rα endows T cells with the ability to compete temporally for limited IL-2 via mechanisms beyond ligand affinity. These results suggest that strategies to enhance IL-2Rα expression on tumor-reactive lymphocytes may facilitate the development of more effective IL-2-based therapies.

BackgroundAdministration of recombinant G-CSF following cytoreductive therapy enhances the recovery of myeloid cells, minimizing the risk of opportunistic infection. Free G-CSF, however, is expensive, exhibits a short half-life, and has poor biological activity in vivo.MethodsWe evaluated whether the biological activity of G-CSF could be improved by pre-association with anti-G-CSF mAb prior to injection into mice.ResultsWe find that the efficacy of G-CSF therapy can be enhanced more than 100-fold by pre-association of G-CSF with an anti-G-CSF monoclonal antibody (mAb). Compared with G-CSF alone, administration of G-CSF/anti-G-CSF mAb complexes induced the potent expansion of CD11b+Gr-1+ myeloid cells in mice with or without concomitant cytoreductive treatment including radiation or chemotherapy. Despite driving the dramatic expansion of myeloid cells, in vivo antigen-specific CD8+ T cell immune responses were not compromised. Furthermore, injection of G-CSF/anti-G-CSF mAb complexes heightened protective immunity to bacterial infection. As a measure of clinical value, we also found that antibody complexes improved G-CSF biological activity much more significantly than pegylation.ConclusionsOur findings provide the first evidence that antibody cytokine complexes can effectively expand myeloid cells, and furthermore, that G-CSF/anti-G-CSF mAb complexes may provide an improved method for the administration of recombinant G-CSF.

Effective T cell therapy against metastatic cancer requires high frequencies of functional tumor-reactive T cells. However, achieving sustained and elevated levels of tumor-reactive T cells has proven difficult and usually requires highly toxic preconditioning of patients with radiation or chemotherapy to destroy host lymphocytes and suppressive cells. Using the mouse CD8+ T cell pmel-1 TCR transgenic model, we found that the combination of 1) in vitro IL-12-priming of donor T cells during activation and 2) the administration of IL-2/anti-IL-2 mAb complexes after adoptive transfer promoted the dramatic and systemic increase in tumor-reactive T cells in the absence of host lymphodepletion. Importantly, this combinatorial therapy led to the cure of over half of mice with established subcutaneous B16 tumors. Interestingly, while IL-2/anti-IL-2 mAb complexes synergized with ex vivo IL-12-priming, IL-15/sIL-15Rα-Fc complexes failed to mediate comparable synergistic activity despite signaling through the shared IL-2Rβγ subunits. Given that IL-12 priming strongly promotes the upregulation of the high affinity IL-2 receptor subunit, IL-2Rα, our results suggest that selective IL-2Rα engagement uniquely promotes T cell persistence and anti-tumor efficacy in the absence of lymphodepletion. Our findings underscore the importance of T cell polarizing conditions in dictating the requirement of adoptively transferred T cells for exogenous cytokine.