TAP-dependent/-independent priming of CD8 T cells by TAAs

We initially analyzed the Cr-1 antigen (Cr-1 or TDGF-1; www.uniprot.org/uniprot/P51865) that is expressed during embryonic development in men and mice and re-expressed in KPC-derived PDACCs (online supplementary figure S1b,c) and in about 44% of human PDAC.21 22 This antigen contains a single Cr-1:K^b/cr_16-24 epitope located at the extreme COOH-terminus of its ER-targeting SP (SP_Cr-1),22 which is released during Cr-1 maturation by ER-resident SPase (figure 1A). To determine TAP-dependent/-independent presentation of the Cr-1:K^b/cr_16-24 epitope, we used a DNA immunization approach. For this, we generated a Cr-1-encoding vector (pCI/Cr-1; figure 1B) and determined Cr-1 expression in transiently transfected HEK-293 cells. Endoglycosidase H treatment of cell lysates showed that Cr-1 was mainly expressed in an about 20 kDa glycosylated _gpCr-1 form (figure 1C), confirming an almost quantitative SP_Cr-1-mediated translocation of Cr-1 into ER/Golgi compartments. A single injection of pCI/Cr-1 efficiently primed Cr-1:K^b/cr_16-24-specific CD8 T cells in B6 and TAP-deficient (TAP1^-/-) mice, detectable in liver and spleen by ex vivo staining with Cr-1:K^b/cr_16-24 dimers or after peptide-specific stimulation and determination of IFN- γ ⁺ Cr-1:K^b/cr_16-24-specific CD8 T-cell frequencies by FCM (figure 1D; groups 2). Priming of Cr-1:K^b/cr_16-24-specific CD8 T cells thus did not require TAP-mediated antigen processing.

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Figure 1

TAP-independent induction of Cr-1:K^b/cr_16-24-specific CD8 T cells by DNA immunization. (A) Map of the Cripto-1 (Cr-1) protein. The NH₂-terminal signal peptide (SP_Cr-1), the epidermal growth factor (EGF)-like domain, the Cripto_Frl-1_Cryptic domain (CFC) and the glycosylphosphatidylinositol anchor (GPI) are indicated. The amino acid sequences of SP_Cr-1, SP_Cr-1/R26P, the K^b/cr_16-24 epitope and the predicted SPase cleavage site are shown. (B) Schematic presentation of pCI/Cr-1, pCI/Cr-1_R26P (Cr-1 containing an arginine to proline exchange at position 26) and of pCI/stT77Cr-1 (Cr-1 containing a strep-tag (ST) and Hsp73-capturing SV40 T1-77 (T77) domain). (C) lysates of non-transfected HEK-293 cells and HEK-293 cells transiently transfected with pCI/Cr-1 (lanes 1), pCI/Cr-1_R26P (Lane 2) or pCI/stT77Cr-1 (lanes 3) were subjected to SDS-PAGE followed by beta-actin-, Cr-1- or st-specific Western blotting. Where indicated, cell lysates were treated with endoglycosidase H (E-Hf +) or remained untreated (E-Hf -) prior to SDS-PAGE. The positions of glycosylated (gp) and non-glycosylated forms of Cr-1 are indicated. (D) B6 mice were immunized with pCI (group 1, n=6, in two independent experiments), pCI/Cr-1 (group 2, n=6, in two independent experiments), pCI/Cr-1_R26P (group 3, n=3, one experiment) or pCI/stT77-Cr-1 (group 4, n=3, one experiment) and TAP1^-/- mice (n=6, in two independent experiments) were injected with PCI (group 1) or pCI/Cr-1 (group 2). Frequencies of dimer⁺ K^b/cr_16-24 specific CD8 T cells in liver and spleen of immunized mice were determined ex vivo by FCM at day 12 postinjection. Additionally, antigen-specific IFN-γ⁺ CD8 T cells were determined by ex vivo stimulation of spleen cells with K^b/cr_16-24 (closed circles) and control K^b/ova_257-264 peptides (open circles). Graphs show the actual proportions (%) of K^b/cr_16-24-dimer⁺ or IFN-γ⁺ cells of total CD8⁺ T cells±SD for statistical evaluation, indicated groups of mice were compared using one-way ANOVA (with 95% CI) followed by Tukey’s multiple comparisons test (B6) or by the unpaired Student’s t-test (TAP^-/-). Only statistically significant differences are indicated (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). ANOVA, analysis of variance; Cr-1, Cripto-1; FCM, flow cytometry; IFN-γ, interferon-γ; SPase, signal peptidase; SP, signal peptide; TAP, transporter associated with antigen processing.

It has been reported that introduction of a proline (P) immediately following the SP cleavage sites (position +1) generated non-cleavable antigen variants.34 The SP identification program SignalP V. 5.0 (http://www.cbs.dtu.dk/services/SignalP/data.php)35 predicted a cut at position 26 of the murine SP_Cr-1, generating an epitope-containing SP_1-25 fragment (figure 1A). To confirm the crucial role of ER-resident SPase for priming Cr-1:K^b/cr_16-24-specific CD8 T cells, we generated a pCI/Cr-1_R26P vector that contained an amino acid exchange at Cr-1₂₆ from arginine (R) to proline (P) (figure 1A,B). This vector expressed an about 22 kDa gpCr-1_R26P glycoprotein that migrated above the gpCr-1 protein band in SDS-PAGE, demonstrating that the SP_Cr-1 SP was not cleaved off in this protein (figure 1C, lane 2). As a consequence, we could not induce K^b/cr_16-24-specific CD8 T cells in B6 mice by pCI/Cr-1_R26P immunization (figure 1D, group 3). Expression/processing of Cr-1 in the ER of antigen-presenting cells (APCs) targeted by DNA immunization was, thus, a prerequisite to induce Cr-1:K^b/cr_16-24-specific CD8 T cells. Confirmatory, targeting Cr-1 to the cytosol, using a NH₂-terminal, Dna-J homologous SV40/T₇₇ domain that stably binds cytosolic Hsp73 in transfected cells36 37 (pCI/stT77Cr-1) (figure 1B,C; online supplementary figure S2) did not induce Cr-1:K^b/cr_16-24-specific CD8 T cells in vaccinated B6 mice (figure 1D; group 4).

Next, we searched for an epitope-containing tumor antigen that, in contrast to Cr-1, is expressed in the ER of PDACCs but processed for MHC-I presentation and CD8 T cell priming in a TAP-dependent manner. We identified the glycoprotein 70 (gp70), originating from an endogenous murine leukemia virus integrated as a provirus in the mouse germline DNA (www.uniprot.org/uniprot/P03386), which fulfilled these criteria. Gp70 was stably expressed in PDACCs (online supplementary figure S1b,c) but not in somatic cells.23 30 38 In contrast to Cr-1:K^b/cr_16-24, the gp70:K^b/p15E epitope is not located in the ER-targeting SP (SP_gp70) (figure 2A) and its processing/presentation apparently depends on proteasomal processing39 and TAP.38 Therefore, this antigen/epitope needs retrotranslocation from the ER to the cytosol and/or ‘cross-presentation’ for efficient MHC-I processing/presentation.20 40 Neither full-length gp70 nor epitope-containing gp70 fragments induced CD8 T cells in H-2^d BALB/c or H-2^b B6 mice.30 41 42 Confirmatory, we here show that an ER-targeted SP_Igκstgp70 antigen, composed of a gp70_327-615 fragment and a NH₂-terminal heterologous Igκ SP sequence (figure 2A,B), did not induce gp70:K^b/p15E-specific CD8 T cells in B6 mice (figure 2C; groups 2). In contrast, a cytosolic Hsp73-bound stT₇₇gp70 fusion antigen (pCI/stT77gp70) (figure 2A,B; online supplementary figure S2), associated with a high cross-priming capacity,37 43 induced high frequencies of gp70:K^b/p15E-specific CD8 T cells in B6 but not in TAP1^-/- mice (figure 2C; groups 3). Priming of gp70:K^b/p15E and Cr-1:K^b/cr_16-24-specific CD8 T cells thus differed substantially, depending on expression of tumor antigens in the ER versus the cytosol (figures 1 and 2; online supplementary table 1). Furthermore, Cr-1:K^b/cr_16-24- and gp70:K^b/p15E-specific CD8 T cells were functional in B6 mice and almost quantitatively and specifically eliminated CFSE-labeled APCs pulsed with Cr-1:K^b/cr_16-24 (online supplementary figure S3) or gp70:K^b/p15E peptides30 in in vivo cytotoxicity assays. This suggested that priming of these TAA-specific CD8 T cells in B6 mice was not restricted by central and peripheral tolerance mechanisms.

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Figure 2

TAP-dependent induction of gp70:K^b/p15E-specific CD8 T cells by DNA immunization. (A) Schematic presentation of the gp70 protein and its variants stgp70 (a 289-residue gp70_327-615 fragment with a NH₂-terminal strep-tag domain), SP_Igκstgp70 (stgp70 with an additional NH₂-terminal ER-targeting Igκ signal peptide (SP_Igκ)) and stT77gp70 (stgp70 with a 77-residue Hsp73- binding T77 domain between ST and gp70). The position of the K^b/p15E epitope is indicated. (B) Cell lysates of nt HEK-293 cells or cells transiently transfected with pCI/stgp70 (lanes 1), pCI/SP_Igκstgp70 (lanes 2) or pCI/stT77gp70 (lanes 3) were subjected to SDS-PAGE followed by anti-st (upper panels) and anti-beta-actin (lower panels) specific Western blotting. Prior to SDS-PAGE, cell lysates were treated with endoglycosidase HF (E-Hf +) or remained untreated (E-Hf -). The positions of glycosylated (gp) and non-glycosylated forms of gp70 are indicated. (C) B6 mice (upper panels, data from two independent experiments) and TAP1^-/- mice (lower panels, one experiment) (n=3–6) were immunized with pCI (group 1), pCI/SP_Igκstgp70 (group 2) or pCI/stT77gp70 (group 3). Twelve days postimmunization, frequencies of K^b/p15E-dimer⁺ CD8 T cells in spleen and liver were determined by FCM. Additionally, antigen-specific IFN-γ production of CD8 T cells was determined by ex vivo stimulation of spleen cells with K^b/p15E (closed circles) and control K^b/ova_257-264 peptides (open circles). Graphs show the actual proportions (%) of K^b/p15E-dimer⁺ or -IFN-γ⁺ CD8 T cells of total CD8 T cells±SD for evaluation of the statistical significance of differences between indicated groups, one-way ANOVA (with 95% CI) followed by Tukey’s multiple comparisons test was used. Only significant differences are depicted (**p<0.01, ***p<0.001, ****p<0.0001). ANOVA, analysis of variance; Cr-1, Cripto-1; ER, endoplasmic reticulum; FCM, flow cytometry; IFN-γ, interferon-γ; TAP, transporter associated with antigen processing.

Manipulation(s) of KPC cells for their usage as cell-based vaccines

A major aim was to explore whether tumor cell vaccines could be used to prime anti-tumor-specific CD8 T-cell responses. We, thus, asked if MHC-I expression can be restored on KPC tumor cells and affect priming of TAP-independent (Cr-1:K^b/cr_16-24) or TAP-dependent (gp70:K^b/p15E) CD8 T-cell responses, and if tumor cell-initiated immune-suppressive (eg, PD-L1-mediated) mechanisms interfere with these immune responses.

In line with established data, we could upregulate MHC-I expression on different KPC cells by an in vitro treatment with IFN-γ44 (figure 3A). Microarrays performed on untreated and IFN-γ-treated KPC cells identified 291 significant differentially expressed probesets (Benjamini-Hochberg adjusted p<0.05). Among those probesets, 289 probesets (corresponding to 238 genes) were upregulated and only one gene (two probesets) was downregulated in IFN-γ-treated cells (figure 3B; online supplementary table 2). Unsupervised hierarchical clustering-based heatmap of the differentially expressed genes showed clear segregation of untreated and IFN-γ-treated cells (figure 3B). Furthermore, among 91 genes present in the KEGG pathway ‘Antigen processing and presentation’ (mmu04612) 27 genes were significantly upregulated; for example, TAP1 and TAP2 (figure 3C). In line with these findings, Gene Set Enrichment Analysis based on the REACTOME database showed significant upregulation of 105 gene sets in IFN-γ-treated cells (FDR<0.05) including ‘Antigen processing and presentation’ and ‘IFN-γ signaling’ (FDR q value<0.001; p<0.001) (online supplementary figure S4a). In particular, the upregulation of TAP1 was found within the top three leading-edge genes based on the immunological signatures database (online supplementary figure S4b). However, these analyzes also revealed a significant upregulation of coinhibitory PD-L1 (CD274) in IFN-γ-treated KPC cells (online supplementary table 2), correlating with a prominent upregulation of PD-L1 on the cell surface (figure 3A).

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Figure 3

IFN-γ-induced gene expression in KPC cell lines. (A) Different KPC cell lines were either untreated or treated with 20 ng/mL recombinant mouse IFN-γ for 20 hours, followed by FCM analysis of H2-K^b and PD-L1 surface expression. One representative histogram per cell line out of three independent experiments is shown. (B) Unsupervised hierarchical clustering based on differentially expressed probesets by comparing five IFN-γ-treated (for 20 hours) KPC cell lines (+IFN-γ) to the respective untreated KPC cell lines (ctrl). Red represents upregulated probesets while blue shows downregulated ones in the IFN-γ-treated (+IFN-γ) group. The samples clearly clustered based on the experimental arms (pink=ctrl; light blue=+IFN-γ). (c) KEGG ‘Antigen processing and presentation’ (mmu04612) gene expression profile in IFN-γ treated KPCs (+IFN-γ) to untreated KPCs (ctrl). We identified 27 genes that showed significant differential expression (BH adjusted p<0.05) and are listed to the right of the heatmap. FCM, flow cytometry; IFN-γ, interferon-γ.

KPC7735 cells and IFN-γ-pretreated KPC7735 cells efficiently grafted as solid tumors in transplanted B6 mice (figure 4A). This suggested that upregulation of co-inhibitory PD-L1 on IFN-γ-treated KPC7735 cells was sufficient to prevent CD8 T cell-mediated rejection of transplanted tumors through direct activation of the PD-L1-signaling axis with PD-1⁺ CD8 T cells. To confirm this assumption, we transplanted KPC7735 cells into B6 mice and treated them with anti-PD-1 antibody. Anti-PD-1 but not isotype control antibodies inhibited the outgrowth of KPC7735 tumors in three out of six animals (figure 4B), indicating that transplanted KPC7735 cells per se induced CD8 T cells that unleashed their effector functions only after blocking PD-L1/PD-1 interactions.45 Confirmatory, untreated KPC7735 cells and, with a higher robustness, IFN-γ-treated KPC7735 cells induced an antitumor immunity that rejected tumor transplants in PD-1^-/- mice (figure 4C). Comparable to B6 mice, neither non-treated nor IFN-γ-treated KPC7735 cells were rejected in PD-L1^-/- mice (figure 4A–D). We, thus, concluded that PD-L1 molecules on the surface of KPCs function as a dominant gatekeepers that suppressed effector CD8 T-cell responses in B6 mice via the PD-L1/PD-1 signaling axis.

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Figure 4

PD-1/PD-L1-dependent growth of KPC7735 tumors in transplanted hosts. (A) 2.5×10⁵ untreated (left panel, n=9) or IFN-γ treated (+IFN-γ) (right panel, n=8) KPC7735 cells were transplanted subcutaneously into the left flank of B6 mice. Data shown were collected from two independent experiments. (B) B6 mice were transplanted with untreated KPC7735 cells and injected intraperitoneally with anti-PD-1 (n=6) or isotype control antibodies (n=5) on days 3 and 6 post tumor inoculation. (C, D) Untreated (left panels) or IFN-γ treated KPC7735 cells (right panels) were transplanted subcutaneously into PD-1^-/- (C) (n=6 per group) or PD-L1^-/- (D) mice (n=4 per group). Tumor rejection rates are indicated. Data shown were collected from two independent experiments. (E) (Upper panel) in two independent experiments, RAG1^-/- mice (n=8) were transplanted subcutaneously with 2.5×10⁵ naïve KPC7735 cells and tumor development was monitored. (Lower panels) surface expression of MHC class I molecules (H2–K^b) and PD-L1 was assessed on ex vivo isolated KPC7735 tumors at day 12 post-transplantation into B6 or RAG1^-/- mice. One representative histogram (representing an individual explanted tumor cell suspension out of four B6 and RAG1^-/- mice, respectively) is shown. IFN-γ, interferon-γ.

KPC7735 cells transplanted subcutaneously into B6 mice showed a prominent upregulation of both, MHC-I and PD-L1 molecules on the cell surface, resembling the effects of the in vitro IFN-γ-treatment (figure 4E). The upregulation of MHC-I and PD-L1 cell surface expression was weakened in subcutaneous KPC7735 tumors developing in T- and B cell-deficient syngeneic RAG1^-/- mice (figure 4E). This indicated that transplanted KPC7735 cells stimulated the adaptive immune system in immune-competent mice, which in turn induced both, MHC-1 and PD-L1 expression on these tumor cells,5 and thereby established tumor-eradicating effector CD8 T-cell responses when the PD-L1/PD-1 signaling axis was silenced/blocked in anti-PD-1-treated B6 or in PD-1^-/- mice.

To proof that tumor cells induce PD-L1/PD-1-sensitive effector CD8 T cells, we generated PD-L1-deficient KPC7735^PD-L1KO cells using the CRISPR/Cas9 system. KPC7735^PD-L1KO cells upregulated expression of MHC-I but not PD-L1, Cr-1 or gp70 on IFN-γ-treatment (online supplementary figure S5a‒c) and were quantitatively rejected in transplanted B6 mice (figure 5A). An additional challenge of these KPC7735^PD-L1KO-immune, tumor-free B6 mice at day 28 with KPC7735 cells resulted in a quantitative rejection of these transplanted cells (figure 5A) along with a prominent CD8 T-cell response against the TAP-dependent gp70:K^b/p15E but not the TAP-independent Cr-1:K^b/cr_16-24 epitope (figure 5B). Similarly, a single immunization of B6 mice with KPC7735^PD-L1KO cells induced high frequencies of gp70:K^b/p15E-specific CD8 T cells and low frequencies of Cr-1:K^b/cr_16-24-specific CD8 T cells (figure 5C). Interestingly, also KPC7735 cells primed gp70:K^b/p15E- and Cr-1:K^b/cr_16-24-specific CD8 T cells in B6 mice, but the frequencies were lower than in KPC7735^PD-L1KO-immune mice (figure 5C). It was striking that a significant higher proportion of gp70:K^b/p15E-specific CD8 T cells primed in KPC7735-immune B6 mice expressed the PD-1 exhaustion marker (figure 5D), which could limit their effector phenotype and explain the rejection of KPC7735^PD-L1KO but not of KPC7735 tumor cells in these mice.

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Figure 5

Testing the antigenicity of KPC7735^PD-L1KO cells. (A) 2.5×10⁵ KPC7735^PD-L1KO cells were transplanted subcutaneously into the left flank of B6 mice (n=6, from two independent experiments) and tumor growth was monitored. At day 28, mice were challenged with 2.5×10⁵ KPC7735 cells and rejection rates are indicated. (B) Non-treated B6 mice (group 1, n=3) and three tumor-free mice shown in (A) were sacrificed at day 45 (group 2) and Cr-1:K^b/cr_16-24- and gp70:K^b/p15E-specific CD8 T-cell frequencies were determined in the liver and spleen. Graphs show the actual proportions (%) of K^b/p15E-dimer⁺ and K^b/cr_16-24-dimer⁺ cells among all CD8 T cells±SD. Statistical significant differences were determined using the unpaired Student’s t-test. (C) B6 mice were either non-treated (group 1, n=4) or immunized with 2.5×10⁵ KPC7735 (group 2, n=4) and KPC7735^PD-L1KO cells (group 3, n=5). Twelve days post cell injection Cr-1:K^b/cr_16-24- and gp70:K^b/p15E-specific CD8 T-cell frequencies were determined as described in (A). ANOVA, followed by Tukey’s multiple comparisons test, was used to compare T-cell frequencies between all groups. (D) Expression of PD-1 was assessed on gp70:K^b/p15E-dimer⁺ CD8 T cells and statistically evaluated using the unpaired Student’s t-test. In the right panels one representative dot blot, showing the staining of PD-1 on gated K^b/p15E-dimer⁺ CD8 T cells is depicted. Data shown in (C, D) were obtained from one experiment. (*p<0.05, **p<0.01). ANOVA, analysis of variance; Cr-1, Cripto-1; ns, not significant.

Development of a novel IFN-γ-treatment protocol to selectively restore MHC-I presentation-competence on PDACCs

Considering, that the deletion of PD-L1 in tumor cells with the CRISPR/Cas9 system is an attractive but laborious and time-consuming way to generate effective cell-based tumor vaccines for PD-1-competent B6 mice, we explored conditions to manipulate KPC tumor cells for selectively restoring their MHC-I presentation competence. To determine whether the kinetics of IFN-γ-inducible MHC-I and PD-L1 surface expression differ, and could be exploited to manipulate the upregulation of PD-L1, tumor cells were treated for 20 hours with IFN-γ, washed and further cultured in vitro (figure 6A,B). This showed that upregulation of PD-L1 in different KPC cells was less sustained as compared with MHC-I and declined toward basal levels around day 3 (d3) post IFN-γ-treatment start, whereas MHC-I expression still was prominent (figure 6B). In contrast, the expression of gp70 and Cr-1 tumor antigens was not (or rarely) affected by the IFN-γ-treatment protocol (online supplementary figure S6).

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Figure 6

Development of a tumor cell-based immunization protocol. (A) Schematic presentation of the IFN-γ-treatment protocol: KPC7735 cells were treated with IFN-γ for 16–20 hours, washed and further cultured up to day 3 post treatment start. Subsequently, cells were irradiated and subcutaneously transplanted (2.5×10⁵ cells per mouse) into the left flank (L) of mice. After 14–19 days, mice were challenged with 2.5×10⁵ naïve KPC7735 cells into the opposite flank (R). (B) Five different KPC cell lines were treated with 20 ng/mL IFN-γ for 20 hours, washed and further cultured up to 7 days. Surface expression of MHC-I molecules (H2-K^b, open circles) and PD-L1 (closed squares) was assessed at the indicated time points by FCM. (C) B6 mice were injected with irradiated KPC7735 cells (KPC7735-ir, n=5), IFN-γ-treated KPC7735 cells irradiated immediately after the IFN-γ treatment (KPC7735-I-ir, n=5) or IFN-γ-treated KPC7735 cells irradiated at day 3 post-IFN-γ treatment start (KPC7735-Id3-ir, n=4) and challenged at day 19 post immunization with KPC7735 cells. Parallel groups of KPC7735-Id3-ir-immune mice (n=4) were additionally treated with anti-CD4 (n=4) or anti-CD8 antibodies (n=5) on days 3 and 6 post tumor challenge (indicated by an arrow). Graphs show tumor growth on the left (L) and right flank (R) and tumor rejection rates are indicated. Data shown were obtained from one experiment. (D) Similarly, PD-1^-/- mice were injected with KPC7735-ir (n=4) or KPC7735-Id3-ir cells (n=4), challenged at day 19 with KPC7735 cells and tumor growth was monitored. (E) B6 (n=7) and PD-1^-/- mice (n=6) were injected with 2.5×10⁵ IFN-γ-treated KPC-Id3-ir cells in two independent experiments. On day 12 postimmunization, Cr-1:K^b/cr_16-24- and gp70:K^b/p15E-specific CD8 T-cell frequencies were determined in the liver and spleen. Graphs show the actual proportions (%) of K^b/p15E-dimer⁺ and K^b/cr_16-24-dimer⁺ cells among all CD8 T cells±SD, corrected for the frequencies detected in non-treated mice. The two mouse groups were compared using the unpaired Student’s t-test. FCM, flow cytometry; IFN-γ, interferon-γ; ns, not significant.

For immunization studies, we additionally irradiated tumor cells and transplanted either non-treated irradiated KPC7735 cells (KPC7735-ir), KPC7735 cells irradiated immediately after IFN-γ treatment (KPC7735-I-ir) and KPC7735 cells irradiated at d3 post IFN-γ treatment (KPC7735-Id3-ir) into the left flank of B6 mice (figure 6A–C). We did not observe outgrowth of tumors in these mice, confirming the efficacy of our irradiation conditions (figure 6C). At d19 postvaccination, mice were challenged with 2.5×10⁵ naïve KPC7735 cells into the opposite right flank (figure 6A–C). KPC7735-ir and KPC7735-I-ir cells did not induce protective immune responses and consecutively transplanted KPC7735 cells formed solid tumors (figure 6C). In contrast, KPC7735-Id3-ir cells induced a tumor-specific immunity that efficiently suppressed tumor development of transplanted KPC7735 cells (figure 6C). Suppression of tumor growth in this group was reverted by the injection of anti-CD8 but not anti-CD4 mAbs (figure 6C), demonstrating that CD8 T cells played a crucial role in the rejection of KPC7735 tumor transplants. Similarly, KPC7735-Id3-ir cells primed a tumor-protective immunity in PD-1^-/- mice (figure 6D). However, in contrast to B6 mice, vaccination of PD-1^-/- mice with KPC7735-ir cells was also sufficient to suppress the outgrowth of consecutively transplanted KPC7735 cells (figure 6C,D). This suggested that the immune-mediated upregulation of MHC-I and PD-L1 (figure 4E) was, at least transiently active in transplanted KPC7735-ir cells and allowed priming of an anti-tumor response in PD-1^-/- (lacking the PD-L1/PD-1 signaling axis) but not in B6 mice. Confirmatory, irradiation of KPC cells resulted in a prominent cell death (>90%) during a subsequent in vitro culture for 48 hours, but did not significantly affect the IFN-γ-induced upregulation of MHC-I and PD-L1 molecules on the small population of live cells (online supplementary figure S7).

KPC7735-Id3-ir cells primed a prominent gp70:K^b/p15E-specific CD8 T-cell response and a weak Cr-1:K^b/cr_16-24-specific CD8 T-cell response in B6 mice (figure 6E; online supplementary table 1). Interestingly, gp70:K^b/p15E- and Cr-1:K^b/cr_16-24-specific CD8 T-cell frequencies were comparable in KPC7735-Id3-ir-immune B6 and PD-1^-/- mice (figure 6E), indicating that a missing PD-1 expression did not affect de novo priming of CD8 T cells. However, as compared with KPC7735-ir cells that did not elicit a tumor-protective immunity in B6 mice (figure 4C), KPC7735-Id3-ir cells induced higher frequencies of gp70:K^b/p15E-specific CD8 T cells associated with a decreased single positive (PD-1⁺ or LAG-3⁺) and double positive PD-1⁺/LAG-3⁺ exhaustion marker profile (online supplementary figure S8) which could preserve their expansion and/or functional effector phenotype.46 The IFN-γ-treatment protocol could also be used in B6 mice to prime a tumor-specific immunity against different PD-L1⁺/MHC^lo PDACCs (KPC7612-Id3-ir) (online supplementary figure S9). Interestingly, upregulation of PD-L1 in IFN-γ-treated PD-L1⁺/MHC^int MC38 colon adenocarcinoma cells was less sustained as compared with the inducible MHC-I expression and MC38-Id3-ir but not MC38 or MC38-ir cell-based vaccination induced a protective immunity in B6 mice that rejected a subsequent transplant of MC38 cells (online supplementary figure S10).

Above analyzes revealed clear differences in the priming of Cr-1:K^b/cr_16-24- and gp70:K^b/p15E-specific CD8 T-cell responses by DNA-based and cell-based immunization (online supplementary table 1). We, thus, tested whether DNA immunization could be used to complement the TAP-dependent gp70:K^b/p15E-specific CD8 T-cell immunity induced by KPC tumor cell-vaccines. Indeed, coimmunization of B6 mice with pCI/Cr-1 and KPC7735^PD-L1KO cells efficiently primed both, Cr-1:K^b/cr_16-24- and gp70:K^b/p15E-specific CD8 T-cell responses and the respective CD8 T-cell frequencies were even comparable to those primed by individual vaccines (online supplementary figure S11). This strongly suggested that de novo primed Cr-1:K^b/cr_16-24- and gp70:K^b/p15E-specific CD8 T cells did not interfere.