Antibodies which activate the T-cell co-stimulatory receptor 4-1BB or block the co-inhibitory receptor CTLA-4, have demonstrated broad anti-tumor effects but cannot induce rejection of the poorly immunogenic B16-BL6 melanoma without the aid of additional therapeutic interventions 
. In contrast to the more immunogenic B16-F10 model 
, we show that α4-1BB mediates few rejection of the highly aggressive B16-BL6 melanoma even in conjunction with GVAX(). As we have observed previously for combination blockade of CTLA-4 and PD-1, we find that co-administration with an autologous tumor vaccine expressing Flt3-ligand allows these antibodies to co-operate in rejecting more than 50% of pre-implanted B16-BL6 melanomas 
. Using this system, we sought to understand the impact on tumor-specific T-cell responses of simultaneously removing a major brake on expansion via blockade of the co-inhibitory receptor CTLA-4, while at the same time actively driving proliferation and survival through activation of the co-stimulatory receptor 4-1BB.
4-1BB expression is more pronounced on CD8 versus CD4 T-cells and for this reason most tumor immunity studies have focused on the impact of α4-1BB on this subset 
. We found that 4-1BB activation promoted much stronger CD8 infiltration of tumors (), inflammatory cytokine production by peripheral CD8 cells (), and proliferation of tumor infiltrating CD8s (). While CD4 effector infiltration and proliferation were only increased by CTLA-4 blockade (), α4-1BB did increase inflammatory cytokine production from these cells both in the periphery and within the tumor itself. The mechanisms underlying this uncoupling of the proliferative and TH1 cytokine promoting effects of 4-1BB agonist antibody treatment in CD4 versus CD8 cells are of interest to us and will be addressed in future studies.
The activation of CD8 T-cells by α4-1BB coupled with the expansion of CD4 effector T-cells by αCTLA-4 clearly accounts for some of the observed synergy between these agents in rejecting B16 melanomas. 4-1BB activation has been described as a powerful promoter of TH1-type cytokine production 
. In prior studies using the B16-Ova model, we have observed a tendency of co-inhibitory blockade to increase TH2 responses in the vaccine draining lymph node 
. In this case, however, α4-1BB polarized cytokine production to TH1 and induced TNF-α production by CD8 T-cells and IFN-γ production by CD4 cells to levels that exceeded the sum of each individual therapy (). Similarly, combination therapy additively increased the percentage of IFN-γ,TNF-α double producing CD4+ effectors in the tumor (). A portion of the anti-tumor effect of CTLA-4 blockade derives from inhibition of Tregs 
; however, the effects of α4-1BB on Treg proliferation and suppression remain unclear 
. Here, α4-1BB dampened Treg proliferation (Figure S6
), reduced the Treg fraction of TIL (), counteracted αCTLA-4's expansion of absolute Treg numbers in the tumor (Figure S2
), and decreased CTLA-4 and PD-1 expression by Tregs (). We conclude that at least a portion of the benefit of combination co-stimulatory modulation is due to reduction of Treg suppression in the tumor.
Distinct from other co-stimulatory antibodies, we found that α4-1BB induced striking upregulation of KLRG1 on CD8+, and to a lesser degree CD4+, effector T-cells in the tumor ( and ). This KLRG1 upregulation appeared unique to 4-1BB agonist antibody, as we did not observe a similar phenotype in response to αCTLA-4 (), αPD-1, or αPD-L1 (Figure S9
). We found that mice receiving the therapeutically more effective αCTLA-4/α4-1BB combination therapy had 1.7-fold more CD4+KLRG1+ cells infiltrating their tumors relative to mice treated with α4-1BB alone (), suggesting a possible functional significance to this population. In addition, the KLRG1+ fraction of TIL seemed to increase over time for both CD8+ and CD4+ effector T-cells suggesting either enhanced infiltration, survival, or proliferation of these cells (). The phenotype of these KLRG1+ tumor-infiltrating T-cells and their contribution to tumor rejection is of intense interest to us and will be the focus of future study.
The co-inhibitory receptor PD-1 which we have previously shown to be induced on T-cells following CTLA-4 blockade 
, also appears highly induced by 4-1BB activation (). Like CTLA-4, PD-1 can attenuate effector T-cell proliferation and effector function, causing anti-tumor immune responses to fail. The data presented here suggests that combining antibody blockade of PD-1 or both CTLA-4 and PD-1 with activation of 4-1BB may provide substantial benefit to auto-tumor immune responses.
Although our focus was on understanding the mechanisms underlying the therapeutic efficacy of combination αCTLA-4/α4-1BB treatment in the context of FVAX, we did explore potential reasons for the failure of GVAX to support synergy between these antibodies (). While ratios of CD8 T-cells to Tregs within tumors of treated mice were slightly lower in the context of GVAX versus FVAX, the overall pattern of CD8 and CD4 effector T-cell to Treg ratios was similar in both treatment settings (Figure S3
). Combination therapy generated higher fractions of both KLRG1+ CD8+ and CD4+ effector T-cells in the context of FVAX versus GVAX which may be significant in explaining the lack of cooperativity observed with GVAX (Figure S7
). Also, it appeared that with GVAX the fraction of KLRG1+ CD4+ FoxP3- cells might be reduced in the combination treated mice relative to the mice receiving α4-1BB alone (mean of 31% vs. 23%); however, the magnitude of this difference did not reach statistical significance for the number of mice analyzed. We also found that although the numbers of KLRG1+ effectors per mm3 of tumor were higher with combination treatment versus α4-1BB alone, the numbers of highly-suppressive KLRG1+ Tregs were also increased (Figure S8
). In addition to this increase of KLRG1+ Treg cells, we found far less advantageous effector T-cell to MDSC ratios in the tumors of mice treated with GVAX as opposed to FVAX (). Also, the higher CD4+ to MDSC ratios observed with FVAX and αCTLA-4/α4-1BB versus α4-1BB alone were not evident in the context of GVAX. This increased suppressive burden in the context of GVAX versus FVAX could be reducing the fraction of tumor-infiltrating effector T-cells which develop into optimal effectors as well as reducing the cooperative augmentation of effector function we observe with FVAX.
Blockade of the co-inhibitory receptor CTLA-4 in the clinic has shown promise as a therapy for advanced solid tumors, however immune related adverse events associated with this treatment can be quite severe 
. The major side of effect of 4-1BB agonist antibody treatment, at least in mice, appears to be an as yet poorly mechanistically defined inflammatory liver toxicity 
. Interestingly, it has been reported in a pre-clinical model that αCTLA-4 and α4-1BB each reduced the auto-reactive side effects of the other 
. Taken with our data showing strong cooperativity between these agents in rejecting B16-BL6 melanoma, further studies are certainly warranted to assess the safety of using 4-1BB agonist antibody and CTLA-4 blockade in combination.