mainly because indicated in the number legends (20?g per RNA in 200?l if not otherwise stated).43?The TLR7 agonist SC1 was injected Teglarinad chloride directly into the tumor (80.5?g in 30?l) at day time 14, Furin 19, 24 and 28 after tumor inoculation. result in anti-tumoral activity despite induction of specific T cells. This is to our knowledge the first statement that neoepitope specific CD8+ T cells primed by tumor-released antigen exposure can be functionally irrelevant. by IFN ELISpot and determine against which naturally processed and offered point-mutated antigens T Teglarinad chloride cells were prevalent (Number 1(a)). Open in a separate window Number 1. Treatment of CT26 bearing mice having a TLR7 agonist induces a discrete neoepitope C specific T-cell response against mutated Smc3. A: Design of the peptide matrix encoding all 628 transcribed non synonymous single nucleotide variants (nsSNVs) of CT26. B: Splenocytes were isolated from CT26-WT tumor bearing mice (n?=?3, 28?days after tumor inoculation, mean tumor size ~800mm3). 5??105 CD4 depleted cells per well were tested for recognition of matrix peptides or 5??104 CT26-WT cells in an IFN ELISpot. C-D: CT26-WT (C) or CT26-gp70KO (D) tumor bearing mice (n?=?5) were treated repetitively with SC1, an TLR7 agonist injected into the tumor starting at day time 14 (tumor size ~50 mm3). T-cell reactions were analyzed by ELISpot on day time 31 as explained above. E-F: Splenocytes from TLR7 treated CT26-WT (E) or CT26-gp70KO (F) tumor bearing mice were tested for acknowledgement of Smc3 and gp70 AH1 peptides at 0.4?g/ml (same concentration as compared to the peptide matrix) or 2?g/ml as well while CT26-WT or CT26-gp70KO cells by IFN ELISpot. Mean + s.e.m. of duplicates is definitely demonstrated. First, we tested for spontaneously occuring point-mutation specific CD8+ T cells in untreated mice bearing subcutaneous (s.c.) CT26 tumors. Splenocytes were harvested 28?days after the mice were inoculated with CT26 tumor cells (CT26-WT) and tumors had reached a mean size of ~800 mm3. CD4+ T cell depleted splenocytes were tested in IFN ELISpot for acknowledgement of the peptide matrix swimming pools. IFN secretion by CD8+ T cells co-cultured with CT26-WT cells was very low and none of the point mutations was specifically recognized (Number 1(b)). We only recognized a T-cell response against the H2-Ld restricted epitope SPSYVYHQF (also called AH1?) of gp70, a well-known non-mutated immunodominant epitope derived from an endogenous retrovirus (Supplementary Number 1),18?which is the highest expressed gene in CT26.19 Having demonstrated the lack of spontaneously happening neoepitope specific T cells in this mouse tumor model, we hypothesized that we could broaden the repertoire of tumor-directed T-cell responses by increasing tumor cell death and thereby antigen release in the context of immunomodulation. To this end we carried out three series of experiments in which tumor-bearing mice were treated with either a TLR7 agonist, were vaccinated in combination with local irradiation or were treated with an anti-PD-L1 antibody for immune checkpoint blockade. SC1, a novel TLR7 agonistic small molecule, is definitely reported to induce potent and durable T cell-mediated tumor control and inflammatory switch of the tumor microenvironment,20,21manuscript in preparation. To ensure adequate antigen exposure and time for priming of T cells, we treated mice 14?days after tumor inoculation when tumors reached a size of 50mm3 with intratumoral (i.t.) Teglarinad chloride injection of SC1. On day time 31 after tumor inoculation, about two weeks after starting treatment, CD4+ T cell-depleted splenocytes were tested for acknowledgement of the peptide matrix swimming pools. Two peptide swimming pools, 17 and 29, were shown to induce IFN secretion above background levels (Number 1(c)). Both swimming pools contained modified peptides derived from a mutant Smc3 (Structural maintenance of chromosomes 3) D733A neoepitope. Smc3 encodes a nuclear protein involved in mitosis that can be secreted after post-translational changes. Overexpressed22?but not mutated Smc3 was described to be involved in tumorigenesis suggesting the detected D733A alteration is a passenger mutation. To exclude the highly abundant gp70 epitope would inhibit the induction of diversified CD8+ T-cell reactions23?we used the CRISPR/Cas9 system to generate a gp70 deficient variant (CT26-gp70KO), which was no longer identified by gp70 AH1-specific CD8+ T cells (Supplementary Number 2). T cells from CT26-gp70KO tumor bearing mice treated with the TLR7 agonist on day time 14 (tumor size ~50mm3) still only recognized the two swimming pools comprising the Smc3 Teglarinad chloride peptides (Number Teglarinad chloride 1(d)). Solitary peptide testing confirmed the induced T cells were.