Introduction

Immunotherapies such as immune checkpoint blockade (ICB) have reshaped the cancer treatment landscape, inducing long-term remissions in a subset of patients.1 In contrast, while vaccines have been enormously successful in combating infectious disease, vaccines for non-viral cancers have had more limited success.2 Most preclinical and clinical studies on tumor-specific CD8 T cell (TST) vaccine responses have been done in the context of established/late tumors.3 Less is known about how TST respond and differentiate in response to immunotherapy during early stages of tumorigenesis when few cells harboring oncogenic mutations and potential neoantigens are present.4

We previously developed an autochthonous mouse model of liver cancer (AST;Cre-ERT2) in which we could initiate liver carcinogenesis with tamoxifen (TAM)-induced Cre-mediated SV40 large T antigen (TAG) expression in hepatocytes.5 TAG acts both as an oncogene, driving liver carcinogenesis, and as a tumor-specific neoantigen recognized by CD8 T cells; this model allows precise temporal control of the duration of TST interactions with transformed hepatocytes and tumors. However, in contrast to human tumors, which arise sporadically and progress clonally,4 TAM-induced oncogene induction is highly efficient, resulting in high antigen burden even in early-stage lesions. To better model sporadic cancer formation and subsequent progression, we allowed AST;Cre-ERT2 mice to undergo stochastic TAG oncogene activation through sporadic, TAM-independent Cre-mediated activity.

We found that TST in AST;Cre-ERT2 mice with early lesions upregulated PD1 and proliferated. Progenitor PD1+TST which express the transcription factor (TF) TCF1 and are localized in secondary lymphoid organs or tertiary lymphoid structures have been shown to maintain the TST population and mediate immunotherapy responses6–9 and reviewed in Philip and Schietinger and Tooley et al.10 11 In mice with early lesions, we found that a subset of PD1+ TST expressed the TF TCF1 and retained the ability to produce IFNγ. This subset was not sustained in mice with late lesions. Here, we tested whether immunization and/or ICB could improve TST function and found that immunization but not ICB generated a robust population of functional TST, which was able to halt and prevent long-term cancer progression in liver cancer-prone mice.

Materials and methodsMice

All mice were bred and maintained in a specific pathogen-free barrier facility at Vanderbilt University Medical Center. TCRTAG transgenic mice (Strain 005236), Cre-ERT2 (Strain 008463), C57BL/6 (Strain 000664), and C57BL/6J Thy1.1 mice (Strain 000406) were purchased from The Jackson Laboratory. TCRTAG mice were crossed to Thy1.1 mice to generate TCRTAG;Thy1.1 and TCRTAG;Thy1.1/Thy1.2 (Thy1.12) mice. AST ((Albumin-floxStop-SV40 large T antigen) mice, obtained from Drs. Natalio Garbi and Günter Hämmerling, German Cancer Research Center and previously described,12 were crossed to Cre-ERT2 mice to generate AST;Cre-ERT2.

Tumor cohort setup

Age-matched and sex-matched AST;Cre-ERT2 were assigned to experimental tumor cohorts at 6 weeks of age. Both male and female mice were used for experimental cohorts. All tumor cohort mice were monitored twice weekly for body condition score (BCS) and abdominal distension (scored from 1 to 4: 1 no swelling, 2 mild abdominal enlargement, 3 moderate abdominal enlargement, 4 marked abdominal enlargement). Mice were analyzed at endpoint or specified time points as specified in figure legends and text. For survival studies, mice were euthanized at prespecified endpoints: (BCS 2), abdominal girth score 4, or difficulty with movement or eating/drinking. For intervention studies (Listeria monocytogenes (LM) vaccination and ICB), mice in AST;Cre-ERT2 tumor cohorts were randomly assigned to treatment arms. Sample size was determined to obtain 80% power to detect a 35% difference between groups, based on our previously observed/published differences and reproducibility. Potential confounders such as animal/cage location, treatment order, and measurement order were minimized by co-housing mice receiving different treatments. CRDR and MP were aware of the group allocation throughout experiments.

Liver tissue preparation, fixing and paraffin block

Liver tissue was removed from AST;Cre-ERT2 mice and immediately transferred into 10% buffered formalin for a minimum of 48 hours before transfer and storage in 70% ethanol. The Vanderbilt Translational Pathology Shared Resource performed paraffin embedding as follows: 70% ethanol for 30 min, 80% ethanol for 45 min, 95% ethanol for 45 min (twice), 100% ethanol for 45 min, 100% ethanol for 35 min (twice), xylene for 35 min (twice), wax (paraffin) for 60 min (three times).

Immunohistochemistry

SV40 TAG-specific mouse monoclonal antibody (BD Bioscience 554149) primary antibody was used. Antigen retrieval was performed with pH 6.0 citrate buffer at 105°C pressure cooker for 15 min and a 10 min bench cool down. Mouse on Mouse Ig Block was performed with Vector MKB-2213 and incubated for 60 min. Peroxidase block was performed at 0.03% H202 w/sodium azide and incubated for 5 min. Primary antibody was diluted at 1:800 and incubated for 60 min. Detection was performed with Dako EnVision+System HRP Labeled Polymer and incubated for 30 min. Chromogen was performed with DAB+ and incubated for 5 min.

Digital image analysis

Digital analysis of whole slide images was performed at the Digital Histology Shared Resources (VUMC). Whole slide images were captured on an Aperio AT2 slide scanner (Leica). Scripts were developed to quantify the number of TAG immunopositive (+) cells per tissue. The levels of DAB chromogen were extracted by color unmixing13 from these whole slide images. Unmixed images were then processed with machine learning using the Ilastik software package14 to produce probability maps of positive signal. Cells were identified and filtered by their size, location, and presence of a detectable nucleus. Positive cell counts were calculated per total number of cells and per tissue area.

Cell isolation

Spleens were mechanically disrupted to a single-cell suspension with the back of a 3 mL syringe plunger, passed through a 70 µm strainer and lysed with ammonium chloride potassium (ACK) buffer (150 mmol/L NH4Cl, 10 mmol/L KHCO3, 0.1 mmol/L Na2EDTA). Cells were washed once and resuspended with RPMI-10: RPMI 1640 (Corning MT10040CV) supplemented with 10% FBS (Corning 35010CV). Livers were mechanically disrupted to a single-cell suspension using a glass pestle against a 150 µm metal mesh in cold PBS containing 2% FBS (2% FBS) and filtered through a 100 µm strainer. The liver homogenate was spun down at 400 g for 5 min at 4°C, and the pellet was resuspended in 15 mL 2% FBS, 500 U heparin (NDC 63323-540-05), and 10 mL PBS Buffered Percoll (Cytiva 17089102), mixed by inversion, and spun at 500 g for 10 min at 4°C. Pellets, enriched for liver-infiltrating lymphocytes, were lysed with ACK buffer, and cells were resuspended in RPMI-10 for downstream analysis. Periportal and celiac lymph nodes were collected and pooled for liver-draining lymph node (ldLN) analysis. Lymph nodes were mechanically dissociated into single-cell solutions using the textured surface of two frosted microscope slides into cold RPMI-10. Isolated leukocytes from spleen, lymph nodes, and liver were analyzed by flow cytometry as described below.

Adoptive T cell transfer

To transfer naive TCRTAG T cells into AST;Cre-ERT2mice, bulk splenocytes were sterilely isolated and processed from TCRTAG;Thy1.1 or TCRTAG;Thy1.12 transgenic mice as described above. To determine the number of CD8+Thy1.1+ or Thy1.12+TCRTAG, an aliquot was taken from the single-cell splenocyte suspension after ACK lysis and counted via hemocytometer to obtain total splenocyte concentration and stained with antibodies for CD8, Thy1.1 and Thy1.12 Splenocytes were then resuspended in final volume of 200 uL of RPMI and injected intravenous into each recipient mouse. For experiments focused on early TST time points (60 hours to 21 days), 2.5×106 CD8+ TCRTAG;Thy1.1 and/or TCRTAG;Thy1.12 were injected per mouse in order to obtain sufficient numbers of cells across different cell divisions. For later immunization experiments where the goal was to reconstitute AST;Cre-ERT2 with a low number of naive TAG-specific T cell, we transferred 0.5×106 TCRTAG;Thy1.1/mouse.

Carboxy fluorescein succinimidyl ester labeling

Splenocytes were isolated and processed as described above and resuspended in 2.5 mL of RPMI. They were then rapidly mixed with equal volume of 2×carboxy fluorescein succinimidyl ester (CFSE) (10 µM) solution and incubated for 5 min at 37°C at a final CFSE concentration (5 µM). Stained cells were quenched by mixing with 5 mL pure FBS, washed twice with RPMI, and resuspended in 200 µL of RPMI for injection.

Listeria infection

LM ΔactA ΔinlB expressing Tag-I epitope (SAINNYAQKL, SV40 large T antigen206–215) (LMTAG),5 or without exogenous antigens (LMΦ), were generated by Aduro Biotech and stored at −80°C. Mice were infected with 5×106 c.f.u. in 200 µL total volume of LMTAG or LMΦ via intravenous injection.

Immune checkpoint blockade

Anti-PD1 (clone RMP1-14) and anti-PDL1 (clone 10.F.9G2) antibodies or isotype control (clone LTA-2) were purchased from BioXcell. Antibodies were diluted in 1X sterile PBS and injected in 200 µL total volume via i.p. injection every other day for five doses, at 200 µg per antibody per mouse.

Antibodies and reagents

Fluorochrome-conjugated antibodies and cell dyes were purchased from Miltenyi Biotech, Thermo, BioLegend, Tonbo/Cytek Biosciences, and Cell Signaling Technology. Specific antibodies are listed in table 1, and cell dyes are listed in table 2.

Table 1

Flow cytometry antibodies

Table 2

Flow cytometry cell dyes

Splenocytes or liver-infiltrating lymphocytes from naive TCRTAG mice or AST;Cre-ERT2 mice were stained with Ghost Dye Red 780 Viability Dye (1:2000 dilution) Tonbo/Cytek 13-0865 T500) and antibodies against surface molecules (CD8, Thy1.1, Thy1.12, CD44, PD1, LY108) in 2% FBS. Flow cytometry plots shown in figures are gated on live CD8+Thy1.1+ or Thy1.12+transgenic TCRTAG. The gating strategy is shown in online supplemental figure 1. For intracellular staining, splenocytes or liver-infiltrating lymphocytes were surface stained as above and then fixed and permeabilized using the FoxP3 TF Fix/Perm (Tonbo/Cytek TNB-0607) per the manufacturer’s instructions before staining for intracellular molecules (TCF1, TOX, KI67). The samples were then analyzed by flow cytometry (see Flow cytometric analysis). For analysis of effector cytokine production (IFNγ, TNFα), ex vivo peptide stimulation was performed prior to staining. Bulk splenocytes or liver lymphocyte fractions were obtained as described above from naive or tumor-bearing AST;Cre-ERT2 mice and mixed with 2×106 C57BL/6 splenocytes and incubated in RPMI-10 for 4 hours at 37°C in the presence of brefeldin A (Biolegend 420601) and TAG peptide (SAINNYAQKL (0.5 µM); Genscript; custom-synthesized). The cells were then surface-stained (CD8, Thy1.1, Thy1.12), fixed and permeabilized as described above, stained with antibodies against IFNγ and TNFα and analyzed by flow cytometry.

Flow cytometric analysis

Flow cytometric analysis was performed using an Attune NxT four laser Acoustic Focusing Cytometer (ThermoFisher). Flow data were analyzed with FlowJo V.10 software (BD Biosciences).

Statistics

Survival curves were generated using the Kaplan-Meier method and compared using the log-rank (Mantel-Cox) test. For comparison of multiple survival curves, significance threshold was set using Bonferroni correction. For comparisons between two groups, Student’s t-test was performed. For multiple comparisons, one-way or two-way analysis of variance (ANOVA) was performed, followed by either post hoc Tukey or Šidák test. The alpha level was set at 0.05. GraphPad Prism V.10 was used for all statistical analyses.

ResultsAST;Cre-ERT2 mice sporadically develop liver tumors which progress with age

We previously developed an autochthonous mouse model of liver cancer (AST;Cre-ERT2) in which we can study TST interactions with cancer cells throughout carcinogenesis.5 In AST;Cre-ERT2 mice, activation of Cre recombinase by a single dose of tamoxifen (TAM) induces expression of the SV40 large T antigen (TAG) under control of the albumin promoter/enhancer in hepatocytes. AST;Cre-ERT2 develop large liver tumors within 60–80 days post-TAM. To study tumor-specific CD8 T cell (TST) responses against TAG-driven tumors, we used congenic donor lymphocytes from transgenic mice in which CD8 T cells express a single T cell receptor (TCR) specific for TAG epitope-I (TCRTAG).15 We found that TST became dysfunctional in TAM-treated AST;Cre-ERT2 mice and were unable to halt tumor progression.5 However, TAM-treated AST;Cre-ERT2 mice had a substantial tumor antigen burden, even at early stages of tumorigenesis, due to efficient TAM-induced Cre recombination. In most patients, oncogene activation occurs sporadically, thus we sought to determine how TST would differentiate and function in mice with rare sporadic transformed hepatocytes or early liver lesions.

In Cre-ERT2 mice, Cre recombinase can undergo stochastic tamoxifen-independent nuclear translocation,16 17 putting hepatocytes in AST;Cre-ERT2 mice at risk of spontaneous TAG oncogene activation. Indeed, we found that as AST;Cre-ERT2 mice aged, they reproducibly developed liver tumors (figure 1A) and progressed to endpoint within ~225 days (figure 1B), much later than TAM-induced liver carcinogenesis. Liver tumors become grossly visible only after AST;Cre-ERT2 mice were >90 days, and mice >130 days had large tumors (figure 1A). For subsequent studies, we grouped 40–80 days aged mice without macroscopic liver lesions as “early”, 90–120 days age mice with small visible tumors as “intermediate”, and >130 days aged mice with large tumors as “late” liver lesion time points (figure 1A). We performed immunohistochemistry staining for TAG on livers from early, intermediate, and late AST;Cre-ERT2 mice. We used a machine learning algorithm (Ilastik)14 to aid in identifying nuclei with positive signal, enabling us to quantify TAG nuclear expression in hepatocytes across scanned whole slide liver images. While early AST;Cre-ERT2 mice had rare small foci of TAG-positive hepatocytes, the percentage of TAG-positive hepatocytes progressively increased with time (figure 1A, right panel). The liver weight of AST;Cre-ERT2 mice increased due to tumor burden, with an initial slow growth phase followed by a more rapid growth phase (figure 1C).

Figure 1

AST;Cre-ERT2 mice sporadically develop liver tumors with age. (A) Upper panel shows the timeline of tumor development in AST;Cre-ERT2 mice, with the middle panel showing corresponding gross liver images (measured in (cm)) and the lower panel showing TAG immunohistochemistry staining. Left, AST;Cre-ERT2 mice aged 40–80 days with grossly normal liver and few TAG+hepatocytes are classed as “early”; middle, aged 90–120 days mice with small macroscopic liver tumors and larger foci of TAG+hepatocytes are classed as ‘intermediate’; right; >130 days mice with large tumors occupying most of the liver and large TAG+lesions are “late.” Representative gross and TAG immunohistochemistry images of early, intermediate, and late AST;Cre-ERT2 mice are shown. Scale bar=50 μ. Right, percentage of total hepatocyte expressing TAG. Each symbol represents an individual mouse with n=3–6/group. ****p<0.0001 (one-way ANOVA with post hoc Tukey test). (B) Kaplan-Meier curve showing overall survival of AST;Cre-ERT2 mice across early, intermediate, and late time points, n=11. (C) Liver weights of AST;Cre-ERT2 male (n=59) mice plotted against age. Black dotted line shows baseline weights of tumor-free B6 and AST mice. ANOVA, analysis of variance.

TST are activated and proliferate in mice with early and late lesions

To compare initial TST differentiation in mice with early versus late liver lesions, we transferred CFSE-labeled naive TCRTAG into early and late time point AST;Cre-ERT2 mice (figure 2A). TCRTAG underwent extensive proliferation in mice with early or late liver lesions, upregulating PD1 as they divided (figure 2B, C). TCRTAG in mice with early lesions divided more slowly, particularly in the spleen and ldLN (figure 2B, C), and there were fewer TCRTAG in the spleens, ldLN, and livers of early mice as compared with late mice (figure 2D). The decreased TST proliferation in mice with early lesions may be due to the lower TAG antigen burden (figure 1A); previous work has shown that TCR signal strength determines the speed and extent of T cell proliferation.18 Within 60 hours of transfer, nearly all TCRTAG in mice with late lesions and most TCRTAG in mice with early lesions failed to produce effector cytokines TNFα and IFNγ (figure 2C). This is in line with our previous studies showing that transferred naive TCRTAG fail to halt tumor progression.5 19 Interestingly, in mice with early lesions, we identified a population of TCRTAG in the spleen and liver that could produce effector cytokines TNFα and IFNγ (figure 2C). Thus, in hosts with sporadic early lesions, a subset of TST resisted rapid differentiation to the dysfunctional state, raising the possibility that this subset might be amenable to immunotherapeutic reprogramming/rescue.

Figure 2

TST activate and proliferate in mice with early and late lesions. (A) Scheme: CFSE-labeled TCRTAG were adoptively transferred into C57BL/6 (B6; naive, gray), early (blue) and late (orange) AST;Cre-ERT2 mice and analyzed 60 hours later. (B) Top, flow analysis of TCRTAG CFSE dilution in the spleens, liver-draining lymph nodes (ldLN) and livers of early, late, and naive mice. This and all subsequent flow plots are gated on live CD8+Thy1.1+ cells (representative gating is shown in online supplemental figure 1A). Data are concatenated from three biological replicates and representative of three independent experiments. Bottom, percentage of TCRTAG in each cell division for spleen, ldLN, and liver. Each symbol represents an individual mouse with n=6–8/group. *p<0.05, **p<0.01, ****p<0.0001 (two-way ANOVA followed by post hoc Šídák test). Biological replicates from two independent experiments were combined. (C) Left, PD1 expression directly ex vivo, and TNFα/IFNγ production after ex vivo TAG peptide stimulation, plotted against CFSE dilution. Naive T cells were assessed only from the spleen and shown as dark gray histograms or dots, naive T cells from the spleen are shown again in light gray in the ldLN and spleen plots for reference. Data are concatenated from three biological replicates and representative of two independent experiments for spleens and livers, and one experiment for ldLN. Right, percentage of TCRTAG positive for PD1, TNFα and IFNγ. Gates for this and subsequent cytokine production figures were set based on no peptide stimulation controls. Each symbol represents an individual mouse with n=7–8/group for spleen and liver and n=3/group for ldLN. ns=not significant, *p<0.05, **p<0.01, ****p<0.0001 (unpaired Student’s t-test). (D) Number of TCRTAG in spleens, ldLN, and livers of early and late mice. Each symbol represents an individual mouse with n=10–13/group combined from three independent experiments for spleens and livers, and n=6 combined from two independent experiments for ldLN. *p<0.05, ***p<0.001, ****p<0.0001 (unpaired Student’s t-test). ANOVA, analysis of variance; CFSE, carboxy fluorescein succinimidyl ester.

A subset of TST remain functional in mice with early liver lesions

To determine whether this functional TST subset persisted, we examined TCRTAG immunophenotype and function 5 days and 21 days post-transfer into early or late AST;Cre-ERT2 mice (figure 3A). As seen at the earlier 60 hours time point (figure 2D), fewer TCRTAG were found in mice with early vs late lesions at 5 days post-transfer, however, the difference became less pronounced at 21 days (figure 3B). TCRTAG in both early and late mice upregulated CD44, indicating antigen exposure and activation20 (online supplemental figure 2). Notably, TCRTAG in early mice continued to express higher levels of PD1 than naive TCRTAG, suggesting that PD1 expression can identify tumor-reactive TST even in hosts with early lesions (figure 3C, upper panels). TST which express the TF TCF1 and localize to secondary lymphoid organs or tertiary lymphoid structures have been associated with improved responsiveness to immunotherapy6–9 (reviewed in Philip and Schietinger and Tooley et al10 11). In contrast, TCF1- TST, predominantly found in the tumor, are resistant to therapeutic reprogramming.19 At 21 days, TCRTAG in early mice expressed higher levels of TCF1 in the spleen than late mice, while in the liver TCRTAG in both early and late mice were TCF1-/low (figure 3C, lower panels) and TOX+ (online supplemental figure 2, lower panels); TOX is a TF associated with terminally differentiated TST.21–25 While nearly all TCRTAG in mice with late lesions were dysfunctional and failed to produce effector cytokines, mice with early lesions retained a subset of IFNγ-producing TCRTAG, even at 21 days (figure 3D). Few TNFα+IFNγ+TCRTAG were found in the livers of mice with early or late lesions, suggesting that TST dysfunction is more severe at the tumor site. These findings are in line with previous work in a mouse model of sporadic cancer arising in all organs which found that early immune tolerance was responsible for tumor progression.26

Figure 3

A subset of TST remain functional in mice with early liver lesions. (A) Scheme: TCRTAG Thy1.1 were transferred into early (dark blue) and late (dark red) AST;Cre-ERT2 mice 21 days prior to analysis, followed by TCRTAG Thy1.12 cells 5 days (light blue, light red) prior to analysis. (B) Number of TCRTAG in spleens and livers of early and late mice. *p<0.05, **p<0.01, ****p<0.0001 (two-way ANOVA with post hoc Tukey test). (C) Left, histograms of TCRTAG PD1 (upper panels) and TCF1 (lower panels) expression. Right, percentage of PD1+ (upper panels) and TCF1+ (lower panels) TCRTAG with positive gate set to exclude (PD1) or include (TCF1) naive TCRTAG (gray). Each symbol represents an individual mouse. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (one-way ANOVA followed by post hoc Tukey test). (D) Left, TCRTAG TNFα and IFNγ production after 4 hours ex vivo TAG peptide stimulation. Right, percentage of TCRTAG positive for IFNγ and TNFα/IFNγ. **p<0.01, ****p<0.0001 (two-way ANOVA, followed by post hoc Šídák test). For B–D, each symbol represents an individual mouse with n=11–13/early group and n=8–9/late group and combined from three independent experiments. ANOVA, analysis of variance.

Vaccination early during tumorigenesis halts cancer progression

We next asked if the functional TST subset present in mice with early lesions could be harnessed to stop tumor progression. LM is a gram-positive intracellular bacterium which induces strong CD4 and CD8 T cell responses.27 Using an actA inlB deficient attenuated LM vaccination strain,28 which has been used in human clinical trials for advanced cancers29–31 and engineered to express the TAG epitope I (LMTAG),5 we tested whether early vaccination of AST;Cre-ERT2 would protect mice from liver cancer progression. Young AST;Cre-ERT2 mice adoptively transferred with naive TCRTAG and the following day were either left untreated (Φ), given a single dose of empty LM (LMΦ), or vaccinated with a single dose of LMTAG and followed, with some cohorts analyzed prior to endpoint (figure 4A). LMTAG-immunization conferred a major survival advantage, with all mice remaining tumor-free and one mouse euthanized for dermatitis without any evidence of liver tumors (figure 4B and online supplemental figure 3A). In contrast, mice in the Φ and LMΦ groups reached endpoint with multiple large liver tumors and increased liver weight (figure 4B, online supplemental figure 3B). At endpoint, most TCRTAG in the LMTAG-immunized mouse made effector cytokines, in contrast to the TCRTAG in the tumor-bearing mice in the Φ and LMΦ groups which were largely unable to produce effector cytokines (online supplemental figure 3C), suggesting that functional TCRTAG prevent liver tumor progression. For these studies, we adoptively transferred a lower number of naive TCRTAG (5×105) for the purpose of reconstituting an endogenous population of TAG-specific CD8 T cells that could be followed longitudinally and targeted with interventions. Accordingly, early AST;Cre-ERT2 that received AT only or LMTAG immunization only did not have improved survival as compared with untreated mice (online supplemental figure 3D), demonstrating that neither TCRTAG nor LMTAG alone were sufficient to halt tumor progression.

Figure 4

Early vaccination prevents liver tumor progression in AST;Cre-ERT2 mice. (A) Scheme: Early AST;Cre-ERT2 mice (42 days) were adoptively transferred with TCRTAG and left untreated (Φ) or the following day, immunized with empty LM (LMΦ) or LM expressing the TAG antigen (LMTAG). One cohort (survival analysis) was followed to humane endpoint and the second (pre-endpoint analysis) analyzed at age 100 days prior to tumor endpoint. The † and numbers indicate the number of mice in the long-term cohort who reached humane endpoint and were euthanized. (B) Kaplan-Meier curve showing survival of mice in each group with n=5–6/treatment group. *p<0.05, **p<0.01 (log rank (Mantel-Cox) test). The ‡ indicates a mouse euthanized for endpoint of severe dermatitis, whereas all other mice were euthanized due to advanced liver tumors. (C–F) Cohort as in A was euthanized and analyzed at age 100 days prior to tumor endpoint. (C) Left, TCRTAG TNFα and IFNγ production after 4 hours ex vivo TAG peptide stimulation. Right, percentage of TCRTAG positive for IFNγ and TNFα/IFNγ. Each symbol represents an individual mouse with n=6–7/group. **p<0.01, ***p<0.001, ****p<0.0001 (one-way ANOVA with post hoc Tukey test). (D) Number of TCRTAG in spleen and livers. Each symbol represents an individual mouse with n=7–9/group. *p<0.05, ***p<0.001 (one-way ANOVA with post hoc Tukey test). (E) Left, histograms of TCRTAG PD1 expression. Right, percentage of PD1+TCRTAG with positive gate set to exclude naive TCRTAG (gray). Each symbol represents an individual mouse with n=2 for naive and n=7–9 for other groups. **p<0.01, ***p<0.001, ****p<0.0001 (one-way ANOVA with post hoc Tukey test). (F) Left, TCF1 and TOX expression in TCRTAG. Right, percentage of TCF1+TOX and TCF1-TOX+TCRTAG. Each symbol represents an individual mouse with n=8–9/group. *p<0.05, **p<0.01, ***p<0.001 (one-way ANOVA with post hoc Tukey test). C–F shows samples pooled from two independent experiments. (G) Early AST;Cre-ERT2 mice (42 days) were adoptively transferred with TCRTAG and immunized with LMTAG 7 days later (early LMTAG) or 60 days later (at age 110 days) (late LMTAG). Kaplan-Meier curve showing survival of mice in each group with n=5–8/treatment group. **p<0.01 (log rank (Mantel-Cox) test). ANOVA, analysis of variance; LM, Listeria monocytogenes.

Progenitor TST are associated with vaccine efficacy

To determine whether the presence of functional TST correlated with later tumor-free survival, we analyzed TCRTAG in Φ, LMΦ, or LMTAG-vaccinated mice at age 100 days, before mice reached endpoint. Most TCRTAG in the spleen and livers of Φ or LMΦ-treated mice failed to make effector cytokines (figure 4C), suggesting that without vaccination, the cytokine-producing TST observed at earlier time points (figure 3D) in mice with early lesions failed to persist long-term. In stark contrast, mice in the LMTAG group had a large subset of double-producing TNFα+IFNγ+ TCRTAG in both the spleen and liver (figure 4C). LMTAG-immunized mice had a larger population of TCRTAG in the spleen (figure 4D), which were PD1− (figure 4E, left panel). Interestingly, there were two populations of TCRTAG in the livers of LMTAG-vaccinated mice, a PD1− and a PD1int population (figure 4E, right panel). LMTAG-vaccinated mice had a higher number of TCF1+TOX-progenitor TST in the spleen and liver as compared with Φ and LMΦ-treated mice (figure 4F). While most TST in LMTAG-vaccinated spleens were TCF1+TOX−, in the liver there were two populations, a TCF1+TOX and a TCF1-TOX+ population (figure 4F, lower panel). Taken together, these data suggest that LMTAG vaccination induces a robust population of functional TST, which sustains long-term antitumor responses, blocking cancer progression.

We next tested whether later vaccination could confer similar protection against liver tumor progression (figure 4G). Immunization at an intermediate time point (100 days), when few progenitor TST exist (Φ group, figure 4F), failed to slow liver tumor progression, and mice succumbed to tumors between 150 and 175 days, suggesting that progenitor TST are required for vaccine antitumor efficacy.

Vaccination is superior to ICB in blocking tumor progression

An important and open question in cancer immunotherapy is how ICB versus vaccination compares in boosting anticancer immune responses, and how best to combine and sequence these therapies.2 32 Therefore, we compared the efficacy of ICB, LMTAG vaccination, and combined ICB/ LMTAG vaccination. Early AST;Cre-ERT2 mice adoptively transferred with TCRTAG and then treated 30 days later with isotype control antibodies (iso), anti-PD1/anti-PD-L1 antibodies (ICB), LMTAG, or combined LMTAG/ICB (figure 5A). ICB conferred no benefit as compared with iso, with all mice developing large liver tumors (figure 5B, C). In contrast, LMTAG and LMTAG/ICB-treated mice had no evidence of tumor progression at 400+ days (figure 5B). We next analyzed TCRTAG in iso, ICB, LMTAG, or ICB/LMTAG-treated mice at age 100 days. LMTAG vaccination, either alone or in combination, led to a substantial increase in TST numbers and IFNγ production, while ICB alone had little impact (figure 5D, E and online supplemental figure 4A,B). Given the complete protection conferred by LMTAG immunization alone, we could not evaluate the therapeutic contribution of ICB. However, the addition of ICB to LMTAG did not improve TCRTAG number or function (figure 5D, E and online supplemental figure 4A,B), even though ICB led to a decrease in PD1 surface staining (figure 5F), and there was a trend toward higher KI67, though not statistically significant (figure 5F). The disconnect between PD1 expression, proliferation, and effector function observed is in line with our previous observations on dysfunctional TST.33 Interestingly, a study of combination therapy in a genetic mouse of pancreatic cancer found that in mice with detectable tumors, LM vaccination alone did not confer protection, while LM followed anti-PD1 ICB 21 days later did improve survival, though most mice eventually succumbed with tumors.34

Figure 5

LMTAG vaccination is superior to immune checkpoint blockade (ICB) in preventing liver tumor progression. (A) Scheme: Early AST;Cre-ERT2 mice (50 days) were adoptively transferred with TCRTAG and treated 30 days later with isotype control antibody, anti-PD1/PDL1 antibodies (ICB), LMTAG, or combination LMTAG/ICB. One cohort (survival analysis) was followed to humane endpoint and the second (pre-endpoint analysis) was analyzed at age 90 days (on completion of ICB). The † and numbers indicate the number of mice in the long-term cohort who reached humane endpoint and were euthanized. (B) Kaplan-Meier curve showing survival of mice in each group with n=3–5/group. *p<0.05, **p<0.01 (log rank (Mantel-Cox) test). (C) Graph showing liver weights at humane end point. Each symbol represents an individual mouse, n=4–5/group. ns=not significant (unpaired Student’s t-test). (D–F) Cohort as in A was analyzed at age 90 days prior to tumor endpoint. (D) Left, TCRTAG from spleen TNFα and IFNγ production after 4 hours ex vivo TAG peptide stimulation. Right, percentage of TCRTAG positive for IFNγ. Each symbol represents an individual mouse with n=2–5/group. *p<0.05, **p<0.01 (one-way ANOVA followed by post hoc Tukey test). (E) Number of TCRTAG in the spleen. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (one-way ANOVA followed by post hoc Tukey test). (F) Left, histograms of TCRTAG PD1 (upper panels) and KI67 (lower panels) expression. Right, percentage of PD1+ (upper panels) and KI67 + (lower panels) TCRTAG with positive gate set to exclude (PD1) or (KI67) naive TCRTAG (gray). Each symbol represents an individual mouse. *p<0.05, ***p<0.001, ****p<0.0001 (one-way ANOVA followed by post hoc Tukey test). ANOVA, analysis of variance; LM, Listeria monocytogenes.

LMTAG immunization elicits a population of progenitor TST

Given that LMTAG immunization and long-term tumor protection was associated with increased numbers of TCF1+TST in the spleen, we next explored the functional properties of these cells and their ability to self-renew and differentiate, the hallmarks of progenitor cells. We adoptively transferred early AST;Cre-ERT2 with TCRTAG and immunized them 40 days later with LMTAG (figure 6A). 60 days later, we sorted LY108+CD8+Thy1.1+TCRTAG from the spleens, transferred them into secondary AST;Cre-ERT2 hosts and analyzed them 8 days later. LY108 is a widely used surrogate marker for TCF1 expression.8 We first examined the correlation between TCF1 expression and functional capacity in the primary immunized hosts and found that cytokine-producing TCRTAG were enriched for TCF1 expressors (figure 6B). Few TCF1- TCRTAG produced cytokine, though not all TCF1+TCRTAG produced cytokine (figure 6B). Thus, TCF1 is not the sole determinant/predictor of function in this context. We next assessed LY108+TCRTAG before and after sorting (figure 6C, D). The pre-sort TCRTAG population in the spleen of LMTAG-immunized mice was heterogeneous for TCF1 and TOX expression, though largely PD1-CD39-. LY108+TCRTAG were uniformly TCF1+and heterogeneous for TOX expression. Eight days later, the progeny of transferred LY108+TCRTAG were found in in higher numbers in the livers of 2° AST;Cre-ERT2 hosts (figure 6C, D). TCF1+TOX-TCRTAG were present in the spleen, and a subset of TCRTAG differentiated to a TCF1-TOX+terminally differentiated state, particularly in the liver. Moreover, TCRTAG in the liver were largely PD1+CD39+, further evidence of tumor-driven terminal differentiation.23 Thus, LY108+TCF1+ TCRTAG elicited through LMTAG immunization in tumor-prone mice behave as functional progenitor TST, capable of self-renewal and terminal differentiation in secondary hosts and protecting against tumor progression.