We have demonstrated that in vivo engagement of OX40 in the context of adoptive transfer of antigen-specific CD4+ T cells is a potent approach that can completely and durably regress advanced B16 melanoma tumors. Our results show that in this context, the OX40 agonist antibody provides superior anti-tumor efficacy compared with other immunomodulatory antibodies when combined with CTX and antigen-specific CD4+ T cells. Under the same conditions, antigen-specific CD4+ T cells achieve higher potency than antigen-specific CD8+ T cells. The efficacy of this therapy was accompanied by increasing Trp1 CD4+ T eff cells and reducing T reg cells, resulting in a favorable T eff/T reg cell ratio within the tumor microenvironment.
Several attempts have been made to engineer T cells from patients in vitro to increase tumoricidal activity. Our results imply that in vivo modulation with antibodies provides an alternative in reprogramming transferred cells to improve clinical outcomes. The availability of immunomodulatory antibodies enhances the feasibility of this approach.
To confirm the clinical applicability of our findings, we established an in vitro system in which the ability of OX40 engagement to increase the tumoricidal capability of human antigen-specific T cells was tested. NY-ESO-1–specific CD4+ T cell lines exposed to a human OX40 agonist increased their ability to lyse autologous melanoma cells, supporting the cytolytic phenotype observed in mouse models.
The selective pressure that the adaptive immune system exerts on tumors leads to antigen down-regulation (Dunn et al., 2002
; Spiotto et al., 2004
). This important escape mechanism poses a challenge when targeting a single antigen. We found, however, that local destruction of tumor cells lacking the target antigen (Trp1) is possible when potent immunotherapies mediate sufficient bystander killing. Although the precise mechanisms leading to the observed effect are currently elusive, it is unlikely that systemic epitope spreading is responsible for the regression of mixed B16:B78H1 tumors because B78H1 tumors were not eliminated when injected concomitantly with B16 at a distant site. We hypothesize that Trp1 cells exposed to OX86 and CTX infiltrate tumors, directly killing antigen-expressing cells and, at the same time, promote a favorable local inflammatory milieu that recruits innate cells eliminating antigen escape variants and/or stromal cells.
Our results indicate that, under CTX-induced lymphopenic conditions, OX40 engagement promotes Trp1 cells as well as endogenous CD4+ T cells to acquire a highly cytotoxic phenotype by inducing Eomes. However, the endogenous CD4+ T cell population is not necessary for eliminating tumors because regression could be recapitulated in mice lacking the capacity to generate adaptive immunity (Rag1−/−). Moreover, the observation that CD4+ T cells with the newly described phenotype have potent direct anti-tumor properties suggests that this approach could be a means to potentiate engineered T cells.
Given that Trp1 cells—in the context of OX40 engagement and lymphopenia—durably control advanced tumors, we further characterized and defined the phenotypic “fingerprint” of these uniquely potent cells. Cytotoxic CD4+
T cells can develop under conditions of chronic antigen exposure leading to a terminally differentiated phenotype (Brown, 2010
). However, upon OX40 stimulation, Trp1 cells not only acquire a terminally differentiated phenotype (Klrg-1high
) but also exhibit markers correlated with memory/low exhaustion (CD62Lhigh
, T-bet low
, and PD-1low
; Belz and Kallies, 2010
; Wherry, 2011
). A recent study showed that Eomes expression enables CD8+
T cells to acquire both effector and memory/self-renewal functions (Banerjee et al., 2010
). An intriguing hypothesis is that the inherent plasticity in both CD4+
T cells allows for the possibility of developing even more potent cells for clinical use when properly stimulated.
The cytokine profile of anti-tumor CD4+
T cells exposed to the combination therapy reveals both Th1 and Th2 cytokine secretion. As opposed to other studies (Qui et al., 2011
) where OX40 and 4-1BB engagement were shown to drive cytotoxic Th1 differentiation, here we show that secretion of both Th1 and Th2 cytokines is a hallmark of our newly described CD4+
T cell population. This difference in CD4+
T cell phenotype may explain the superior anti-tumor efficacy with the conditions described in our manuscript. Moreover, we found that OX86 diminished Th17 and TFh (T follicular helper) lineages, shown by lower levels of IL-17 and down-regulation of Bcl-6 (Johnston et al., 2009
). Although Eomes can inhibit RoRγt, which could account for IL-17 down-regulation (Ichiyama et al., 2011
), the observation that Trp1 cells secrete both Th1 and Th2 cytokines under these conditions is perplexing. Given that T-bet represses Th2 cytokines such as IL-4 to promote Th1 polarization (Zhu et al., 2010
), it is feasible that T-bet down-regulation overcomes IL-4 suppression and high Eomes expression promotes IFN-γ secretion. Moreover, several experiments in viral models where CD4+
T cells were polarized into different helper lineages (Th0, Th1, Th2, and Th17) show that cells with the Th0 phenotype exhibit higher cytotoxic potential (Brown, 2010
). We speculate that OX40 engagement drives Trp1 cells to acquire a highly cytotoxic Th0-like phenotype in vivo, characterized by Eomes expression. This suggests that OX40 engagement may induce a new effector Th lineage which warrants further characterization.
Overall, our observations highlight the clinical potential of OX40 engagement in promoting a cytotoxic CD4+ T cell population with unique phenotypic markers. As adoptive transfer of designer or engineered cells becomes more feasible, optimizing their anti-tumor potential is of clear importance. The ability to modulate cells with sufficient lineage plasticity in vivo with a single monoclonal antibody opens a new avenue and overcomes many of the challenges encountered by adoptive T cell therapy protocols.