In Situ Vaccination Improves Efficacy of PD-1 Blockade in Unresponsive Lymphoma Tumors through Induction of a Highly Efficient Cross-Presenting Dendritic Cell Subset Expressing TLR3

Background

Low-grade non-Hodgkin's B-cell lymphomas are generally incurable, with standard therapies inducing only temporary remissions; and preliminary results with anti-PD-1 therapy have yielded low response rates. It has been shown that tumoral DC infiltration correlates with efficacy of checkpoint blockade. Tumor-targeted vaccines represent promising, novel treatment strategies able to induce anti-tumor T cells. Therefore, we tested the combination of in situ vaccination with PD-1 blockade in a pre-clinical mouse model.

Methods

A20 lymphoma-bearing mice were treated with a PD-1 blocking antibody with or without an in situ vaccine consisting of intratumoral injections of FMS-like tyrosine kinase-3 ligand (Flt3L), local irradiation (XRT) of the tumor and intratumoral injections of the TLR3 agonist poly-ICLC (pIC).

Data

Untreated lymphoma tumors contained very low numbers of DC and treatment with anti-PD1 alone did not induce tumor regression or increase survival. After Flt3L treatment, flow cytometry analysis revealed a dramatic increase of IRF8+TLR3+ DC at the tumor site, the tumor-draining lymph node (TdLN) and the spleen. XRT of A20 cells induced activation of Flt3L-treated splenic DC in vitro and local XRT of the tumor in vivo induced expression of CD103 on infiltrating TLR3+ DC, indicating maturation of these cells. Local XRT also increased the amount of intratumoral DC taking up tumor-associated antigen (TAA) , suggesting enhanced uptake of dying tumor cells. Interestingly, tumor antigens were taken up mainly by CD103+ DC and not CD103- subtypes. CD103+ expression distinguishes a subset of migratory DC that are very efficient at cross-presenting exogenous antigens to CD8+ T cells. Accordingly, CD103+ DC isolated from the tumor induced proliferation of tumor-specific CD8+ T cells more efficiently than CD103- subsets. The combination of Flt3L with XRT and pIC induced tumor-reactive, Interferon γ (IFN γ)-producing T cells, but delayed tumor growth and improved survival only in 40% of mice. Importantly, in situ vaccination also increased expression of PD-L1 on tumor cells and tumor infiltrating DC. Consistent with these findings, combination of in situ vaccination with PD-1 blockade led to complete tumor regression and increased long-term survival in the majority of mice. Moreover, PD-1 blockade also increased the number of tumor-reactive T cells and depletion of CD8+ T cells abrogated the anti-tumor effect.

Conclusions

In situ vaccination combining intratumoral Flt3L injection with local XRT and poly-ICLC can improve efficacy of anti-PD-1 in checkpoint-unresponsive lymphoma tumors through induction of a highly efficient cross-presenting DC subset leading to long-term regression of established lymphoma tumors