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• W4385816229 abstract "Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Cancer immunotherapies, in particular checkpoint blockade immunotherapy (CBT), can induce control of cancer growth, with a fraction of patients experiencing durable responses. However, the majority of patients currently do not respond to CBT and the molecular determinants of resistance have not been fully elucidated. Mounting clinical evidence suggests that the clonal status of neoantigens (NeoAg) impacts the anti-tumor T cell response. High intratumor heterogeneity (ITH), where the majority of NeoAgs are expressed subclonally, is correlated with poor clinical response to CBT and poor infiltration with tumor-reactive T cells. However, the mechanism by which ITH blunts tumor-reactive T cells is unclear. We developed a transplantable murine lung cancer model to characterize the immune response against a defined set of NeoAgs expressed either clonally or subclonally to model low or high ITH, respectively. Here we show that clonal expression of a weakly immunogenic NeoAg with a relatively strong NeoAg increased the immunogenicity of tumors with low but not high ITH. Mechanistically we determined that clonal NeoAg expression allowed cross-presenting dendritic cells to acquire and present both NeoAgs. Dual NeoAg presentation by dendritic cells was associated with a more mature DC phenotype and a higher stimulatory capacity. These data suggest that clonal NeoAg expression can induce more potent anti-tumor responses due to more stimulatory dendritic cell:T cell interactions. Therapeutic vaccination targeting subclonally expressed NeoAgs could be used to boost anti-tumor T cell responses. Editor's evaluation This valuable work explores the influence of intra tumor heterogeneity of neoepitopes within a cancer on the immune response leading to tumor control in vivo using a transplantable murine lung cancer model. It presents convincing evidence that immune responses against weak neoepitopes are enhanced when clonally expressed with strong neoepitopes, due to a more mature DC phenotype and a higher stimulatory capacity of DCs presenting both weak and strong neoepitopes. The work will be of interest to immunologists and cancer immunotherapists. https://doi.org/10.7554/eLife.85263.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Engaging tumor-reactive immune responses has been an incredibly powerful tool in the fight against cancer (Waldman et al., 2020; Esfahani et al., 2020). Cytotoxic CD8+ T cells recognize peptides on class I major histocompatibility complexes (MHCI) expressed on tumor cells, and following recognition mediate specific lysis of their target cell (Stinchcombe et al., 2001; Isaaz et al., 1995). While CD8+ T cells can recognize many tumor-associated antigens, peptides specific to tumor cells are best suited to drive the most powerful anti-tumor responses (Schietinger et al., 2008; Minati et al., 2020). Amongst the tumor-specific antigens, the class of so-called neoantigens (NeoAg) is best understood thus far. NeoAgs are predominantly derived from non-synonymous mutations in highly expressed protein coding transcripts within the tumor cells (Schumacher et al., 2019). It has been shown that patients responding to checkpoint blockade immunotherapy (CBT) often experience an expansion in NeoAg-reactive T cells within tumor-infiltrating T cells, as well as in circulation (van Rooij et al., 2013; Riaz et al., 2017). Further, adoptive cell transfer of NeoAg-specific T cells may be beneficial (Robbins et al., 2013; Verdegaal et al., 2016; Gros et al., 2014) and vaccination can induce objective responses toward tumor-specific NeoAgs (Carreno et al., 2015; Keskin et al., 2019; Johanns et al., 2019; Ott et al., 2017). The presence of CD8+ T cells within the tumor microenvironment (TME) is established as a positive prognostic marker of response to CBT and overall survival (van der Leun et al., 2020). Over the past years, many studies have aimed to establish a correlation between the presence of NeoAgs and CD8+ T cells within the tumor resulting in findings that increased NeoAg burden is positively associated with T cell infiltration in some cancers (Rooney et al., 2015). Despite enormous efforts, several independent reports suggest that NeoAg load alone cannot predict response to CBT (Mauriello et al., 2019; McGrail et al., 2021; Samstein et al., 2019; Ghorani et al., 2018). Of note, it seems the prognostic value of NeoAg burden depends on the baseline presence of a T cell infiltrate (Mauriello et al., 2019; McGrail et al., 2021). This can be best illustrated in cancer types with high mutational burden, such as melanoma, non-small cell lung cancer, and colon cancer. In those cancer types, a sizable proportion of patients lack a productive T cell infiltrate, despite an abundance in predicted NeoAgs (Mauriello et al., 2019; McGrail et al., 2021; Spranger et al., 2016). Past studies have indicated that alterations in tumor cell-intrinsic signaling pathways can mediate poor T cell infiltration, typically by means of poor T cell activation or poor T cell recruitment into the TME (Nguyen and Spranger, 2020; Lawson et al., 2020). However, these alterations do not account for all patients failing to respond to CBT while harboring high numbers of predicted NeoAgs. Recent studies suggest that intratumor heterogeneity (ITH), which might be highest in patients with high mutational burden, impacts the responsiveness to CBT (McGranahan et al., 2016). Clinical data suggest that clonal NeoAg expression is associated with response to anti-PD-1 CBT in a cohort of patients with NSCLC and with significantly increased overall survival in melanoma patients, following treatment with anti-CTLA-4 antibodies (McGranahan et al., 2016). In contrast, subclonal NeoAg expression in tumors with high ITH was found to be associated with poor CBT responses and poor CD8+ T cell infiltration. These observations were confirmed in a transplantable mouse model using subclones derived from a UVB-irradiated murine melanoma cell line (Wolf et al., 2019). While these initial studies strongly suggest that high ITH impairs the anti-tumor immune response, the mechanisms of how the anti-tumor immune response is impaired are still unknown. To interrogate the effect of ITH on anti-tumor T cell responses, we generated a syngeneic transplantable murine lung tumor model that enables us to precisely modulate the degree of ITH using naturally developed NeoAgs. Using two NeoAgs with different degrees of immunogenicity, we elucidated that responses against the weaker NeoAg were potentiated only in the clonal setting. This synergistic effect was established during T cell activation by cross-presenting conventional type I dendritic cells (cDC1), which acquired a more mature phenotype if they presented both antigens. Intriguingly, RNA-based vaccines targeting the weak NeoAg augmented immune responses in tumors with high ITH, highlighting the potential therapeutic value of targeting weakly immunogenic subclonal NeoAgs. Results Cancer cells expressing NeoAgs elicit diverse anti-tumor immune responses Next-generation genome sequencing combined with MHCI binding prediction algorithms and in vivo validation have allowed for the identification of bona fide NeoAgs expressed in murine tumor lines including MC38, B16F10, and TRAMP-C1 (Yadav et al., 2014; Castle et al., 2012; Matsushita et al., 2012). Based on their reported immunogenicity, we selected candidate NeoAgs derived from mutated Adpgk, Aatf, and Cpne1 and immunized C57BL/6 mice with short peptides (8mer and 9mer) containing the mutations to validate their immunogenicity. Immunization with the mutant Adpgk peptide induced appreciable expansion of NeoAg-specific T cells while immunization with Cpne1 and Aatf peptides resulted in low or non-detectable T cell responses, respectively (Figure 1A and Table 1). Figure 1 with 1 supplement see all Download asset Open asset KP6S cell line engineered to express natural neoantigens (NeoAgs) elicit variable anti-tumor immune responses. (A) Mice were vaccinated with short peptides with cyclic-di-GMP as adjuvant. 10 µg of peptide was delivered subcutaneously (s.c.) at the base of the tail along with 25 µg of cyclic-di-GMP. An identical dose was delivered s.c. 10 days following the first dose and spleens were collected at day 21 for IFNγ ELISpot. Quantification of IFNγ-producing cells after restimulation from two independent experiments shown as mean ± SEM (n = 3 per group per experiment). (B) Schematic of the lentiviral construct used to transduce the KP6S subclone. (C, D) Mice were injected s.c. with 1 × 106 tumor cells in (B) WT mice or (C) Rag2-/- mice. Representative data from one of two individual experiments are shown (n = 3 or 4 per group per experiment). Quantification of (E) absolute numbers of CD8+ TIL per gram tumor from six independent experiments (pooled n = 17 per group), (F) proportion of CD8+ TIL at day 9 or 10 after tumor implantation from eight independent experiments (pooled n = 23 per group), (G) proportion of CD44+CD62L- Teffector from eight independent experiments (pooled n = 23 per group), (H) IFNγ-producing cells restimulated 9 or 10 d after tumor implantation using ELISpot from two independent experiments (pooled n = 5 per group). *p<0.05, **p<0.01, ****p<0.0001; one-way ANOVA (Kruskal–Wallis) test in (A), two-way ANOVA (Tukey) in (C, D), Mann–Whitney U in (E–H). Data are shown as mean ± SEM. Figure 1—source data 1 Raw data for Figure 1. https://cdn.elifesciences.org/articles/85263/elife-85263-fig1-data1-v2.xlsx Download elife-85263-fig1-data1-v2.xlsx Table 1 Amino acid sequences of wildtype and NeoAg. NameWildtypeMutantPeptide sequenceSequence positionBinding predictionPredicted affinity (IC50)Peptide sequenceBinding predictionPredicted affinity (IC50)AdpgkASMTNRELM298–307Db6.21ASMTNMELMDb4.29AatfMAPIDHTAM493–501Db297.14MAPIDHTTMDb90.16Cpne1SSPDSLHYL298–307Db764.37SSPYSLHYLDb182.34 We next generated cell lines to assay anti-NeoAg responses in vivo by using KP1233, a lung adenocarcinoma line derived from a KrasG12D/+Trp53−/− mouse (DuPage et al., 2009). Because of the inherent cellular heterogeneity observed in many murine cell lines (Ben-David et al., 2018), we derived a stable subclone, referred to as KP6S, that grew similarly to the parental line in wildtype mice. The clonal KP6S cell line was used exclusively to generate all cell lines used in this study. To drive expression of specific NeoAgs, we expressed one or two NeoAgs linked at the C-terminus of the fluorescent protein mCherry followed by a barcode (Figure 1B). Subcutaneous implantation of the subclones expressing a single NeoAg (KPAdpgk, KPAatf, KPCpne1) indicated that KPAdpgk exhibited early tumor control before growing out while KPAatf and KPCpne1 cell lines grew out progressively (Figure 1C, Figure 1—figure supplement 1A). To confirm that the initial control observed against KPAdpgk tumor cells was mediated by an adaptive immune response, we implanted all cell lines into Rag2-/- mice and observed that all three cell lines grew progressively with similar kinetics (Figure 1D, Figure 1—figure supplement 1B). Naturally arising NeoAgs expressed in human cancer encompass both highly immunogenic and poorly immunogenic sequences. Thus, we chose the Adpgk, Aatf, and Cpne1 NeoAgs to capture the diversity of NeoAg-specific responses observed in humans (Luksza et al., 2017). Analysis of tumor-infiltrating T cells in KPAdpgk and KPAatf showed that KPAdpgk tumors had a greater degree of infiltration with CD8+ T cells compared to KPAatf tumors (Figure 1E and F). Further, CD8+ T cells in KPAdpgk tumors were more activated based on CD44 staining (Figure 1G). Additionally, IFNγ ELISpot showed a greater peripheral expansion of NeoAg-specific T cells in mice implanted with KPAdpgk tumors compared to KPAatf or KPCpne1 tumors (Figure 1H). Assessing the T cell infiltrate in KPCpne1 and KPmCherry tumors revealed that Cpne1-expressing tumor cells were not highly immunogenic as neither CD8+ T cell infiltration nor activation were significantly different between both tumors (Figure 1—figure supplement 1C–E). The immune responses observed corresponded with MHCI binding affinities predicted by NetMHC 4.0 (Nielsen et al., 2003; Andreatta and Nielsen, 2016), with the mutant Adpgk peptide predicted to have an IC50 of 4.29 nM while the other mutant peptides had IC50 values ranging from 90.16 nM (Aatf) to 182.34 nM (Cpne1) (Figure 1—figure supplement 1F). MHCI-stabilization assays also provided evidence that predicted binding affinities captured the range of peptide-MHCI (pMHCI) affinities for our selection of NeoAgs (Figure 1—figure supplement 1G). Thus, we established a model of transplantable syngeneic murine tumor lines that express NeoAgs with varying degrees of immunogenicity. Homogeneous expression of NeoAgs increases the immunogenicity of cancer cells To assess the impact of different NeoAg expression patterns in tumors, we first generated a cell line that expressed both Adpgk and Aatf, hereafter termed KP-HetLow (Figure 2A). To model heterogeneous NeoAg expression patterns (KP-HetHigh), we inoculated C57BL/6 mice with a mixture of 50% KPAatf cells and 50% KPAdpgk cells (Figure 2A). We implanted 1 × 106 cells of KP-HetHigh and KP-HetLow tumors into mice and observed drastically increased control of tumor outgrowth of KP-HetLow tumors compared to single antigen-expressing tumors. In contrast, KP-HetHigh grew progressively, with similar kinetics as observed for KPAatf (Figure 2B). In fact, quantitative PCR analysis of KP-HetHigh tumors showed progressive outgrowth of the KPAatf subclone that completely dominated the tumor by day 14 post-implantation (Figure 2—figure supplement 1A). The control of KP-HetLow tumors was completely lost in Rag2-/- mice (Figure 2B), indicating that tumor control was mediated by an adaptive immune response. We also found that the change in tumor cell composition of KP-HetHigh tumors at later timepoints observed in wildtype mice was also absent in Rag2-/- mutants. Instead, KPAatf and KPAdpgk cells were maintained at nearly a 1:1 ratio (Figure 2—figure supplement 1B), providing evidence for immunoediting in this model. These data indicate that adaptive immune responses are capable of controlling tumors with homogeneous NeoAg expression, whereas tumors with heterogeneous NeoAg expression are characterized by immune editing and escape of non-immunogenic subclones. Figure 2 with 3 supplements see all Download asset Open asset Tumors expressing a pair of neoantigens (NeoAgs) homogeneously have increased immunogenicity. (A) Schematic of the generation of tumors used in (B). (B) Tumor growth of KP-HetHigh and KP-HetLow in WT and Rag2-/- mice. Representative data from one of two individual experiments are shown (n = 3 per group per experiment). (C, D) Splenocytes from tumor-bearing mice were used in an IFNγ ELISpot to determine the frequency of NeoAg-specific T cells in the periphery at days 7, 10, and 14 after tumor implantation. Quantification of the (C) Adpgk-specific response and (D) Aatf-specific response. Pooled data from five independent experiments for day 7 for single antigen tumors and six independent experiments for all other groups (n = 3–4 per group per experiment), four independent experiments for day 10 for single antigen tumors and five independent experiments for all other groups (n = 3 per group per experiment) and three independent experiments for day 14 (n = 3 per group per experiment) in (C, D). (E) Schematic and tumor growth of KPAatf and KPAdpgk in Rag2-/- mice after adoptive T cell transfer (ACT) from naïve or tumor-bearing mice on day 4 after tumor injection. Representative data from one of two individual experiments are shown (n = 4 per group per experiment). *p<0.05, ***p<0.001, ****p<0.0001; two-way ANOVA (Tukey) in (B, E), one-way ANOVA (Kruskal–Wallis followed by Dunn’s multiple-comparisons test) in (C, D). Data are shown as mean ± SEM. Figure 2—source data 1 Raw data for Figure 2. https://cdn.elifesciences.org/articles/85263/elife-85263-fig2-data1-v2.xlsx Download elife-85263-fig2-data1-v2.xlsx To obtain insights into the kinetics of the tumor-reactive T cell response, we assayed NeoAg-specific T cells via IFNγ ELISpot on days 7, 10, and 14 post tumor implantation. The T cell response toward Adpgk was significantly greater in KP-HetLow tumors compared to KP-HetHigh tumors at day 7 and greater than both KP-HetHigh and KPAdpgk tumors at day 10 (Figure 2C). Strikingly, at days 7 and 10, the T cell response against Aatf was only detectable in mice implanted with KP-HetLow tumors and was absent in mice bearing KP-HetHigh or KPAatf tumors (Figure 2D). At day 14, the Aatf and Adpgk responses were similar in all tested conditions, suggesting mixed effects of tumor size, antigen availability, and loss of functional capacity of T cells over time (Figure 2B–D). The observed enhanced T cell response against a weakly immunogenic NeoAg is also observed when Cpne1 was co-expressed with Adpgk (Figure 2—figure supplement 2A and B). We considered the possibility that increasing NeoAg load in a cell could increase immunogenicity by expressing the two weakly immunogenic NeoAgs, Aatf and Cpne1, together. However, this provided no benefit to the Aatf response (Figure 2—figure supplement 3). These data suggest that homogeneous NeoAg expression patterns can increase the peripheral response against poorly immunogenic NeoAgs if they are paired in tandem with a stronger antigen. We next assessed the relative contribution of each NeoAg-specific immune response to the superior control of KP-HetLow tumors. Adoptively transferred CD8+ T cells from KP-HetLow-bearing donor mice slowed the growth of KPAdpgk as well as KPAatf in Rag2-/- mice (Figure 2E). In line with the weaker IFNγ ELISpot responses observed in KP-HetHigh tumors, transfer of CD8+ T cells from KP-HetHigh-bearing donor mice was less beneficial, resulting in improved control of KPAdpgk, but not of KPAatf. While adoptive cell transfer more effectively slowed the growth of KPAdpgk tumors, this suggests that the Aatf-specific immune response contributes to the superior tumor control of KP-HetLow tumors. Batf3+ dendritic cells are required for anti-tumor responses in KP-HetLow tumors Given our observation that peripheral T cell responses against weak NeoAgs are enhanced early following tumor inoculation, we postulated that T cell activation of Aatf-reactive T cells in the lymph node might be different between mice bearing KP-HetLow and KP-HetHigh tumors. While it is established that cross-presenting cDC1 driven by the transcription factor Batf3 are critical for priming CD8+-specific responses (Hildner et al., 2008; Spranger et al., 2015), recent work by us and others have also implicated additional cDC subsets (Duong et al., 2022) or compensatory development of Batf3-independent cDC1 (Tussiwand et al., 2012) in mediating anti-tumor immunity. We thus aimed to determine whether Batf3-dependent cDC1 were required for the increased immune control observed against KP-HetLow tumors. We implanted KP-HetLow tumor cells in wildtype, Rag2-/- and Batf3-/- mice, and observed a loss of tumor control in Rag2-/- and Batf3-/- mice (Figure 3A), indicating that cDC1 are required for the induction of effective T cell responses. Figure 3 with 2 supplements see all Download asset Open asset Cross-presenting dendritic cells mediate the increased immunogenicity of KP-HetLow tumors. (A) Tumor growth of KP-HetHigh tumor cells was implanted subcutaneously (s.c.) into Batf3-/-, Rag2-/- and WT mice. Representative data from three independent experiments (n = 5 per group per experiment). (B) Number of cDC1 in KP-HetHigh and KP-HetLow tumors on days 7, 10, and 14 after s.c. implantation. Pooled data from two independent experiments is shown (n = 3 per group per experiment). (C) Proportion of mCherry+ cDC1 in tumor-draining lymph nodes. Pooled data from two independent experiments for days 7 and 10 and three independent experiments for day 14 is shown (n = 3 per group per experiment). (D) Median fluorescence intensity of the mCherry signal of cells from (B). (E) Experimental schematic for (F, G). Tumor cells were irradiated with 40 Gy and 1.5 × 106 total irradiated cells were immediately s.c. injected into mice. A short peptide boost with both peptides and c-di-GMP as adjuvant was given 10 d after and administered s.c. at the base of the day. 21 days after the irradiated cell implantation, spleens were collected for ELISpot. (F) Peripheral Aatf-specific response. Pooled data from three independent experiments are shown (pooled n = 11 or 12 per group). (G) Peripheral Adpgk-specific response. Pooled data from one or three independent experiments are shown (n = 6 for KPAdpgk and pooled n = 12 for remaining groups). *p<0.05, **p<0.01, ****p<0.0001; two-way ANOVA (Tukey) in (A), Mann–Whitney U for each time point between the two tumors was assessed in (B–D), one-way ANOVA (Kruskal–Wallis followed by Dunn’s multiple-comparisons test) in (F, G). Data are shown as mean ± SEM. Figure 3—source data 1 Raw data for Figure 3. https://cdn.elifesciences.org/articles/85263/elife-85263-fig3-data1-v2.xlsx Download elife-85263-fig3-data1-v2.xlsx cDC1 can impact anti-tumor T cell responses during T cell activation in the tumor-draining lymph node (TdLN) or by facilitating recruitment to the tumor (Spranger et al., 2015). Since we observed differences in CD8+ T cell infiltration between KP-HetLow and KP-HetHigh, we first assessed the number of tumor-infiltrating cDC1. However, while we observed dynamic changes in the absolute numbers of cDC1 over time, no significant difference was found between the two tumor conditions (Figure 3B, Figure 3—figure supplement 1). To track cDC1 carrying tumor cell debris, we controlled for mCherry expression in all cell lines by assessing the fluorescent intensity using flow cytometry to ensure equal antigen and fluorophore expression (Figure 3—figure supplement 2). Assessing the number of tumor cell debris carrying mCherry+ cDC1 in the TdLN further affirmed that the differences in T cell activation were not driven by a lack of migratory cDC1 bringing antigen to the TdLN as similar frequencies were detected between the two tumor conditions (Figure 3C, Figure 3—figure supplement 1). Analysis of the mCherry MFI amongst the mCherry+ cDC1 similarly showed no significant difference between the KP-HetLow and KP-HetHigh conditions (Figure 3D), suggesting that neither cDC1 recruitment to the tumor, trafficking to the TdLN, nor amount of available antigen can explain the observed differences in T cell activation. In the homogeneous KP-HetLow setting, it is conceivable that epitope spreading in response to a rapid and strong Adpgk-specific T cell response might lead to an increase in available Aatf antigen as killing of KP-HetLow cells would result in release of both Adpgk and Aatf-containing debris. This increase in antigen abundance could explain an increase in activation of Aatf-reactive T cells compared to the KP-HetHigh setting. To test whether antigen availability alone might explain the differences in T cell response, we inoculated mice with lethally irradiated tumor cells using single-antigen-expressing tumor cell lines (KPAatf or KPAdpgk), or the KP-HetLow and KP-HetHigh conditions (Figure 3E). To ensure robust responses, we recalled T cell responses with an equal mixture of purified Adpgk and Aatf short peptides combined with cyclic-di-GMP as an adjuvant 10 d after the initial injection of irradiated tumor cells (Figure 3E). Then 11 d post recall, T cell responses were assessed using an IFNγ ELISpot assay (Figure 3E). Consistent with our previous observations, we observed that the Aatf-specific T cell response was dependent on the context of the NeoAg expression patterns, with greater expansion of Aatf-specific T cells in response to KP-HetLow tumor debris compared to either KPAatf or KP-HetHigh tumor debris (Figure 3F). In contrast, we did observe that the Adpgk response was sensitive to lower antigen availability as mice injected with irradiated KP-HetHigh tumor cells, where only 50% of the cells express Adpgk, also exhibited a significantly reduced expansion of Adpgk-specific T cells in the periphery compared to KPAdpgk and KP-HetLow, both tumors where all the cells express Adpgk (Figure 3G). This result is consistent with previous reports on the correlation between antigen availability and strength of T cell response, where providing less Adpgk debris resulted in a corresponding decrease in the Adpgk-specific response (Bullock et al., 2003; Westcott et al., 2021). In sum, we identified that NeoAg expression patterns are critical for priming responses against weak NeoAgs, while the antigen load impacts responses toward strong NeoAgs. NeoAg presentation on dendritic cells mirrors NeoAg expression patterns in the TME It has been shown that the same dendritic cell can take up debris containing both MHCII- and MHCI-restricted epitopes, allowing the DC to interact with CD4+ T cells for licensing to then activate a productive CD8+ T cell response (Ferris et al., 2020b). Similarly, reports suggest that interactions between a DC and CD8+ T cells can impact the maturation state of the DC (Mailliard et al., 2002; Hernandez et al., 2007). We thus considered the possibility that a strong MHCI epitope might act as a ‘licensing’ response to a weaker MHCI epitope when presented on the same DC. To test this notion, we regenerated the KP-HetLow cell lines by expressing Adpgk linked at the C-terminus of ZsGreen (ZsG) while Aatf maintained its expression with mCherry establishing KP-HetLow(ZsG-Adpgk,Aatf), and a corresponding KPZsG-Adpgk as control (Figure 4—figure supplement 1A). We confirmed that these tumor cell lines recapitulated the previously observed outgrowth kinetics (Figure 4—figure supplement 1B). Given that we established the importance of cDC1 for T cell priming, we focused our analysis on this DC subset and used mCherry and ZsGreen as a readout for tumor cell debris engulfment and antigen presentation (Figure 4A). We first determined the proportion of single-fluorophore or double-fluorophore positive cDC1 in the TdLN at day 7 post tumor implantation and found that in KP-HetHigh tumors most of the cDC1 carrying detectable debris were either mCherry+ or ZsGreen+ (Figure 4B). In stark contrast, most tumor cell debris-positive cDC1 found in the TdLN-draining KP-HetLow tumors were double positive for both mCherry and ZsGreen (Figure 4B). Within the single positive cDC1 subset in both tumors, there was a bias toward ZsGreen+ cells (Figure 4C), which could be attributed to the stability of this fluorescent protein (Yi et al., 2022). Figure 4 with 1 supplement see all Download asset Open asset Antigen presentation on dendritic cells in the tumor-draining lymph node mirror antigen expression patterns in the tumor microenvironment (TME). (A) Experimental schematic for (B, C). KP-HetHigh tumors were composed of KPZsG-Adpgk and KPAatf; KP-HetLow tumors were composed of KP-HetLow(ZsG-Adpgk,Aatf). (B) Quantification of the proportion of cDC1 that are double positive (mCherry+ZsGreen+) in tumors. (C) Proportion of mCherry+ or Zsgreen+ cDC1 in the single positive population. Pooled data from three independent experiments are shown (pooled n = 10 per group) for (B, C). (D) Normalized CD40 median fluorescence intensity for single-positive and double-positive populations. (E) Normalized CD80 median fluorescence intensity for the same sample populations in (D). Pooled data from three independent experiments are shown (pooled n = 13 per group) for (D) and (E). *p<0.05, **p<0.01; one-way ANOVA (Kruskal–Wallis followed by Dunn’s multiple-comparisons test) in (B–E). Data are shown as mean ± SEM. Figure 4—source data 1 Raw data for Figure 4. https://cdn.elifesciences.org/articles/85263/elife-85263-fig4-data1-v2.xlsx Download elife-85263-fig4-data1-v2.xlsx Previous reports have indicated that costimulatory markers were upregulated on dendritic cells following ‘licensing’ interactions with CD4+ but also CD8+ T cells (Mailliard et al., 2002; Hernandez et al., 2007; Carenza et al., 2019; Min et al., 2010). We therefore assessed the expression of CD40 and CD80 on single or double fluorophore-positive cDC1 populations in the TdLN. Affirming our initial hypothesis, we observed significantly greater expression of the costimulatory molecules CD40 and CD80 in double-positive cDC1 compared to mCherry+ cDC1 that engulf only Aatf-containing debris (Figure 4D and E). CD40 expression was comparable between Adpgk-ZsGreen+ and double-positive cDC1, while CD80 was highly expressed on both these cDC1 populations (Figure 4D and E), suggesting that the Adpgk-specific T cell response might induce upregulation of co-stimulatory molecules. In sum, these findings suggest that the antigen-dependent interaction between cDC1 and Adpgk-specific T cells could result in increased activation (‘licensing’) of cDC1, and subsequently, an increased capacity of cDC1 to prime Aatf-reactive T cells, if the same cDC1 also presents the weaker NeoAg. Prophylactic RNA vaccination expands Aatf-specific T cells and increases response of heterogeneous tumors to CBT Clinically, a high degree of ITH is associated with poor responses to CBT. To determine whether our established model system faithfully recapitulated resistance to therapy, we inoculated KP-H" @default.
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• W4385816229 title "Decision letter: Decoupled neoantigen cross-presentation by dendritic cells limits anti-tumor immunity against tumors with heterogeneous neoantigen expression" @default.
• W4385816229 doi "https://doi.org/10.7554/elife.85263.sa1" @default.
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