One problem that limits the efficacy of adoptive T cell therapies is rapid tolerization or deletion of transferred tumor-reactive T cells in cancer patients. Although recent studies have shown that younger or central memory-like CD8+
T cells are more potent and persist longer than effector memory-like T cells in the setting of ACT (44
), their increased requirement for costimulatory support may heighten the influence of tolerogenic DCs on these transferred cells. Because of its critical and natural role in DC activation, CD40 ligation has been explored to activate tolerogenic DCs in the tumor environment. Although systemic administration of agonist anti-CD40 antibodies has been shown to replace the need for CD4+
T cell help (12
), boost CD8+
T cell responses to tumors and break peripheral self-tolerance (15
), there is also evidence that it can induce immune suppression (18
). Similarly, we found that systemic administration of an agonist anti-CD40 antibody in TRP-SIY mice initially stimulated an anti-tumor CD8+
T cell response, but eventually led to severe immune suppression, in the context of an influenza infection (). In addition, systemic anti-CD40 administration is associated with significant side effects (data not shown). The complicated and variable outcomes of systemic CD40 ligation in immune responses highlight the need to induce CD40 ligation locally in the tumor tissue.
Here, we report a novel strategy to activate tolerogenic DCs by using tumor-reactive CD8+
T cells to deliver an activating CD40L signal in the tumor environment. This strategy allows us to localize the immunostimulatory signal in both time and space. CD40L is normally expressed on activated CD4+
T cells for less than 24 hrs (11
), and its expression on activated CD8+
T cells is similarly transient. Because CD40L transcription is driven by a retroviral LTR in our study, the tight regulation of CD40L expression on the surface of CD8+
T cells is likely regulated at the post-translational level. Supporting this notion, deletion of the terminal 13 amino acid residues of the cytoplasmic domain led to a higher level and extended duration of CD40L expression on the surface of CD8+
T cells. This deletion mutant is designed to minimize receptor-mediated endocytosis, but does not likely impact other regulatory mechanisms that also control CD40L expression, such as down regulation of CD40L transcription, proteolytic cleavage, and release of soluble CD40 (11
), and thus surface CD40L expression is still transient. We used in vitro memory 2C T cells to deliver CD40L to the correct location, because they recognize a tumor-derived epitope (SIY) and naturally traffic to the TDLNs and tumor tissue. Following adoptive transfer, only those retrovirally-transduced 2C cells that traffic to the PDLN have the opportunity to encounter SIY and re-express CD40L. Even with truncation of the majority of the cytoplasmic domain, CD40L is only expressed on the surface of 2C T cells for 48 to 72 hrs after activation (). Consequently, delivery of the immunostimulatory CD40L signal is limited spatially and temporally. This should minimize the potential autoimmune complications and immune suppression associated with systemic CD40 ligation.
Like CD40L expressed on activated CD4+
T cells, CD40L expressed on the surface of activated CD8+
T cells also stimulates maturation of DCs both in vitro and in vivo (). Co-culture of BMDCs with CD40L-expressing 2C cells for 24 hrs is sufficient to stimulate up-regulation of CD80 and CD86 on DCs, to a similar extent as observed with LPS stimulation (). Similarly, transfer of CD40L-expressing 2C cells into TRP-SIY mice stimulated up-regulation of CD80 and CD86 on some DCs in the PDLN. Despite the modest observable effect on DC maturation in vivo, the anti-tumor CD8+
T cell response was significantly enhanced, as indicated by the increased percentage of reporter 2C cells that could produce IFNγ in the PDLN and infiltrate the prostate tissue. The enhanced anti-tumor response was even more dramatic when the CD40L-expressing 2C cells were activated by an influenza infection, as opposed to by the tolerizing environment of the tumor (). Following influenza infection, a higher fraction of retrovirally-transduced 2C T cells re-expressed CD40L, which simulated maturation of more DCs in the PDLN (Supplemental Fig. 4
). Thus, augmented anti-tumor T cell responses can be induced by engineering T cells to deliver a CD40L-mediated stimulatory signal to dendritic cells in the TDLNs.
There are two critical environments in which tolerance needs to be prevented or broken for adoptive T cell transfer to be most effective: the TDLNs and the tumor itself. If tolerance is broken in the tumor, but not in the TDLNs, adoptively transferred T cells may be tolerized upon initial transfer. This is particularly true for central memory-like T cells that traffic through the secondary lymphoid tissues prior to entering the tumor. If tolerance is broken in the TDLNs, but not in the tumor, adoptively transferred T cells may be tolerized upon entering the tumor tissue. Our approach breaks tolerance in the lymphoid tissues, but not in the tumor itself. As the retrovirus-mediated CD40L expression is transient, the engineered 2C cells no longer express CD40L when they infiltrate the tumor, approximately 7 days post transfer (data not shown, (39
)). As a result, neither dendritic cells nor CD40-expressing tumor cells in the prostate tissue are impacted. Thus, following transfer of CD40L-expressing therapeutic 2C cells, adoptively transferred reporter 2C cells become productively primed in the PDLN, maintain their function in the periphery, infiltrate the tumor tissue extensively, but become rapidly tolerized in the tumor (Supplemental Fig. 3
). Since the tumor-infiltrating T cells become tolerized, we did not evaluate alterations in disease progression or overall survival of treated mice. To overcome tolerance in both the TDLNs and tumor tissue, our approach needs to be combined with other approaches that break tolerance in the tumor tissue. Alternatively, CD40L could be further engineered to achieve more prolonged surface expression.
Recent pre-clinical and clinical studies have made significant progress in advancing the success of adoptive cell therapy in the clinic. T cell engineering strategies may further augment the promise of such approaches. Our study demonstrates that adoptively transferred T cells can be engineered to deliver functional signals to dendritic cells in the tumor environment, and that those dendritic cells can then stimulate more robust T cell responses. As we better understand the critical role that dendritic cells play in the activation, maintenance and tolerization of T cells in the tumor environment, similar approaches can be explored to engineer tumor-reactive CD8+ T cells to deliver important functional signals to dendritic cells to overcome tumor tolerance.