To understand why cancer vaccine-induced T cells often fail to eradicate tumors, we studied immune responses in mice vaccinated with gp100 melanoma peptide in incomplete Freund’s adjuvant (IFA), commonly used in clinical cancer vaccine trials. Peptide/IFA vaccination primed tumor-specific CD8+ T cells, which accumulated not in tumors but at the persisting, antigen-rich vaccination site. Once there, primed T cells became dysfunctional and underwent antigen-driven, Interferon-γ (IFN-γ) and Fas ligand (FasL)-mediated apoptosis, resulting in hyporesponsiveness to subsequent vaccination. Provision of anti-CD40 antibody, Toll-like receptor 7 (TLR7) agonist and interleukin-2 (IL-2) reduced T cell apoptosis but did not prevent vaccination site sequestration. A non-persisting vaccine formulation shifted T cell localization towards tumors, inducing superior anti-tumor activity while reducing systemic T cell dysfunction and promoting memory formation. Persisting peptide/IFA vaccine depots can induce specific T cell sequestration, dysfunction and deletion at vaccination sites; short-lived formulations may overcome these limitations and result in greater therapeutic efficacy of peptide-based cancer vaccines.

Intratumoral immune activation can induce local and systemic anti-tumor immunity. Imiquimod is a cream-formulated, TLR-7 agonist that is FDA-approved for the treatment of non-melanoma skin cancers, but has limited activity against melanoma. We studied the anti-tumor activity and mechanism of action of a novel, injectable, tissue-retained TLR 7/8 agonist, 3M-052, which avoids systemic distribution. Intratumoral administration of 3M-052 generated systemic anti-tumor immunity, and suppressed both injected and distant, uninjected wild-type B16.F10 melanomas. Treated tumors showed increased level of CCL2 chemokines and infiltration of M1 phenotype-shifted macrophages, which could kill tumor cells directly through production of nitric oxide and CCL2, was essential for the anti-tumor activity of 3M-052. CD8+ T cells, B cells, Type I IFN, IFN-γ, and pDC were contributed to efficient tumor suppression whereas perforin, NK cells and CD4 T cells were not required. Finally, 3M-052 therapy potentiated checkpoint blockade therapy with anti-CTLA-4 and anti-PD-L1 antibodies, even when checkpoint blockade alone was ineffective. Our findings suggest that intratumoral treatment with 3M-052 is a promising approach for the treatment of cancer and establish a rational strategy and mechanistic understanding for combination therapy with intratumoral, tissue-retained TLR7/8 agonist and checkpoint blockade in metastatic cancer.

Anticancer vaccination is a promising approach to increase the efficacy of cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed death ligand 1 (PD-L1) checkpoint blockade therapies. However, the landmark FDA registration trial for anti-CTLA-4 therapy (ipilimumab) revealed a complete lack of benefit of adding vaccination with gp100 peptide formulated in incomplete Freund's adjuvant (IFA). Here, using a mouse model of melanoma, we found that gp100 vaccination induced gp100-specific effector T cells (Teffs), which dominantly forced trafficking of anti-CTLA-4-induced, non-gp100-specific Teffs away from the tumor, reducing tumor control. The inflamed vaccination site subsequently also sequestered and destroyed anti-CTLA-4-induced Teffs with specificities for tumor antigens other than gp100, reducing the antitumor efficacy of anti-CTLA-4 therapy. Mechanistically, Teffs at the vaccination site recruited inflammatory monocytes, which in turn attracted additional Teffs in a vicious cycle mediated by IFN-γ, CXCR3, ICAM-1, and CCL2, dependent on IFA formulation. In contrast, nonpersistent vaccine formulations based on dendritic cells, viral vectors, or water-soluble peptides potently synergized with checkpoint blockade of both CTLA-4 and PD-L1 and induced complete tumor regression, including in settings of primary resistance to dual checkpoint blockade. We conclude that cancer vaccine formulation can dominantly determine synergy, or lack thereof, with CTLA-4 and PD-L1 checkpoint blockade therapy for cancer.

CD40 agonists bind the CD40 molecule on antigen-presenting cells and activate them to prime tumor-specific CD8+ T cell responses. Here, we study the antitumor activity and mechanism of action of a nonreplicating adenovirus encoding a chimeric, membrane-bound CD40 ligand (ISF35). Intratumoral administration of ISF35 in subcutaneous B16 melanomas generates tumor-specific, CD8+ T cells that express PD-1 and suppress tumor growth. Combination therapy of ISF35 with systemic anti-PD-1 generates greater antitumor activity than each respective monotherapy. Triple combination of ISF35, anti-PD-1, and anti-CTLA-4 results in complete eradication of injected and noninjected subcutaneous tumors, as well as melanoma tumors in the brain. Therapeutic efficacy is associated with increases in the systemic level of tumor-specific CD8+ T cells, and an increased ratio of intratumoral CD8+ T cells to CD4+ Tregs. These results provide a proof of concept of systemic antitumor activity after intratumoral CD40 triggering with ISF35 in combination with checkpoint blockade for multifocal cancer, including the brain.

Although the synergetic interactions between chemolithoautotroph Acidithiobacillus ferrooxidans and heterotroph Acidiphilium acidophilum have drawn a share of attention, the influence of Aph. acidophilum on growth and metabolic functions of At. ferrooxidans is still unknown on transcriptional level. To assess this influence, a co-culture composed by At. ferrooxidans and Aph. acidophilum was successfully acclimated in this study. Depending on the growth dynamics, At. ferrooxidans in co-culture had 2 days longer exponential phase and 5 times more cell number than that in pure culture. The ferrous iron concentration in culture medium and the expression of iron oxidation-related genes revealed that the energy acquisition of At. ferrooxidans in co-culture was more efficient than that in pure culture. Besides, the analysis of CO2 fixation-related genes in At. ferrooxidans indicated that the second copy of RuBisCO-encoding genes cbbLS-2 and the positive regulator-encoding gene cbbR were up-regulated in co-culture system. All of these results verified that Aph. acidophilum could heterotrophically grow with At. ferrooxidans and promote the growth of it. By means of activating iron oxidation-related genes and the second set of cbbLS genes in At. ferrooxidans, the Aph. acidophilum facilitated the iron oxidation and CO2 fixation by At. ferrooxidans.