The use of ACT with tumor specific T cells has shown to be quite effective for the treatment of established melanoma both in animal models as well as in patients. However, eradication of established melanoma has not been consistently achieved when using T cells specific for naturally-processed, tumor-associated Ags (e.g. gp100) (19
). In the current study, we clearly demonstrate that a myeloablative conditioning plus syngeneic BMT dramatically increased the anti-tumor efficacy of ACT using gp100 specific Tc1 or Tc17 cells against established B16 lung tumors. Using a BMT platform, we further showed that gp100-specific Tc1 cells could eradicate established parental B16 melanoma, and that gp100-spcific Tc17 cells could eradicate established IFNγRDN
B16 tumors. To our knowledge, this is the first report showing that the ACT with T cells specific for naturally-processed tumor-associated Ags completely cured established melanomas without the use of IL-2 or vaccination.
To stringently assess the potency of Tc1 and Tc17 cells in anti-tumor responses, we utilized an established lung-metastatic model of B16 melanoma, a poorly immunogenic (31
) and a highly aggressive tumor in C57BL/6 mice (32
). Analogous to human melanoma, B16 melanoma expresses the mouse homologue (pmel-17) of human gp100 (33
), a naturally processed melanoma-associated Ag. We utilized pmel-1 TCR transgenic T cells specific for gp100 to generate Tc1 and Tc17 cells as effector T cells. Consistent with previous reports that ACT with tumor-specific T cells cannot eradicate established tumors (23
), we observed that either adoptive transfer with pmel-1 Tc1 or Tc17 cells only partially controlled tumor growth in mice that received non-myeloablative conditioning (). However, the therapeutic efficacy of ACT was significantly augmented using Tc1 or Tc17 cells in mice that received myeloablative conditioning followed by TCD-BMT. We reason that myeloablative conditioning and hematopoeitic stem cells increased the expansion and/or infiltration of transferred T cells, as the numbers of Tc1 and Tc17 cells were increased in the spleen and lung of those treated mice ().
Using BMT as a platform, Tc1 cells exhibited anti-tumor activity superior to Tc17 cells (). Effective tumor rejection by adoptively transferred T cells is likely to rely on several direct and indirect immune effector mechanisms. First, both Tc1 and Tc17 effectors may directly eradicate tumor cells through cognate interactions involving perforin/granzyme mediated lytic mechanisms (4
). As a result, tumor-associated Ags may be released and presented by host APCs that may enhance host immune responses at sites proximal and distal to tumor growth. Second, release of Tc1 and Tc17 cytokines, such as IFNγ and TNFα, have been shown to directly inhibit tumor cell growth (34
), as they enhance Ag presentation through up-regulation of MHC-I on both tumors and host APCs (37
), and increase the expression of effector molecules by T cells to facilitate anti-tumor responses and tumor rejection (39
). Third, Tc1 and Tc17 effector cells can induce Ag non-specific inflammatory responses that may indirectly aid in cytolytic and/or cytostatic anti-tumor effects. Local release of cytokines, such as Tc17-derived IFNγ, TNF-α, IL-17 and IL-21 and Tc1-derived IFNγ, have been shown to mediate the selective recruitment and localization of macrophages, NK cells, and granulocytes that may facilitate enhancement of tumor Ag presentation and inhibition of tumor growth (21
). In either case, utilization of discrete cytokines produced by Tc1 or Tc17 effector cells can mediate tumor rejection with distinct mechanisms, and potentially affect the efficacy of anti-tumor responses and tumor regression.
Following ACT, Tc1 cells proliferated substantially faster than Tc17 cells during first 5 days after cell transfer (), but the numbers of Tc17 cells were significantly higher in spleen on days 14 and 28 regardless of tumor types (). We interpret that Tc1 cells have superior potential to proliferate than Tc17 cells, but Tc17 cells have an advantage to survive as supported by our previously published observation (43
). In WT B16 tumor, Tc1 cells were more efficient than Tc17 cells to mount anti-tumor responses against this poorly immunogenic melanoma ( and ). Because the comparable numbers of Tc1 and Tc17 cells were observed at the tumor site (lung) on days 14 and 28, we reason that the therapeutic efficacy of Tc1 and Tc17 cells against B16 tumor did not result from their differential expansion or infiltration in vivo
. Instead, we propose that it is the quality of transferred T cells that dictates the therapeutic efficacy. Tc1 cells are known to have strong cytolytic activity against tumor targets, and the current study demonstrated that IFNγ-signaling through tumor cells was required for Tc1-mediated anti-tumor activity (). Consistent with recent reports showing that Tc17 cells have much weaker cytolytic activity (4
), we also found that Tc1 cells expressed significantly higher levels of CD107a than Tc17 cells () Tc17 cells could produce IFNγ after adoptive transfer in vivo
, but the amount of IFNγ or the frequency of IFNγ-producing cells was lower than that observed in Tc1 cells. As Tc17-mediated anti-tumor activity was also dependent on IFNγ, we surmise that lower cytolytic activity and IFNγ production by Tc17 cells likely accounted for their inferior therapeutic efficacy against B16 tumor as compared to Tc1 cells.
Strikingly and unexpectedly, we found that Tc17 cells were highly effective against IFNγRDN
melanoma (). It appears to be counterintuitive that neutralization of IFNγ not only inhibited Tc17-mediated anti-tumor activity against parental B16 but against IFNγRDN
B16 cells as well. Because IFNγRDN
B16 cells express 10-fold more dominate-negative receptors that cannot mediate IFNγ signaling, it is clear that Tc17-mediated anti-tumor activity is independent of receptor signaling on tumor cells. However, IFNγRDN
B16 cells could absorb significant amount of IFNγ. We surmise that a large number of IFNγ/IFNγR complexes created a microenvironment that promoted Tc17-mediated killing, which was independent of IL-17A since neutralization of this cytokine had no effect (). It is likely that the IFNγ/IFNγR complexes on the IFNγRDN
B16 cells serves as a rich source of IFNγ for the tumor microenvironment, which promotes the anti-tumor effect. Although a precise mechanism remains to be further investigated, additional data ( and ) suggest that abundant IFNγ accumulated at the tumor site might increase the recruitment of Tc17 cells through CXCR3 and the Tc17 activity by enhancing IFNγ+
subset, which has strong anti-tumor activity (44
). In addition, Tc17 cells have the phenotype of long-lived effector T cells (CD127hi
), which may also contribute Tc17-mediated anti-tumor response against IFNγRDN
tumor. Other potential mechanisms may include that other cytokines produced by Tc17 such as IL-21 and IL-22 could exert indirect anti-tumor response against IFNγRDN
tumor by recruiting innate immune cells such as macrophages and neutrophils.
Overall, the current study leads to two novel and important findings. 1) the combination of BMT with TBI and adoptive transfer of tumor specific Tc1 cells can cure established melanoma, which may represent a new strategy to treat patients with metastatic melanoma; 2) local accumulation of IFNγ at the tumor site significantly contributes to T-cell mediated anti-tumor responses independent of IFNγ-signaling by tumor cells, which may have an implication that IFNγ inside the tumor may act as an adjuvant to improve immunotherapy against established tumor.