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Intravenous Nanoparticle Vaccination Generates Stem-Like TCF1+ Neoantigen-Specific CD8+ T Cells

Personalized cancer vaccines are a promising approach for inducing T cell immunity to tumor neoantigens. Using a self-assembling nanoparticle vaccine that links neoantigen peptides to a TLR7/8 agonist (SNP-7/8a), we show how the route and dose alter the magnitude and quality of neoantigen-specific CD8+ T cells. Intravenous vaccination (SNP-IV) induced a higher proportion of TCF1+PD-1+ CD8+ T cells compared to subcutaneous immunization (SNP-SC). Single cell RNA-seq showed that SNP-IV induced stem-like genes (Tcf7, Slamf6, Xcl1) whereas SNP-SC enriched for effector genes (Gzmb, Klrg1, Cx3cr1). Stem-like cells generated by SNP-IV proliferated and differentiated into effector cells upon checkpoint blockade leading to superior antitumor response compared to SNP-SC in a therapeutic model. The duration of antigen presentation by dendritic cells controlled the magnitude and quality of CD8+ T cells. These data demonstrate how to optimize antitumor immunity by modulating vaccine parameters for specific generation of effector or stem-like CD8+ T cells.


INTRODUCTION
The clinical effectiveness of cancer immunotherapy, including checkpoint blockade and adoptive T cell therapy, is based on the magnitude, quality, breadth and ability of T cells to infiltrate tumors [1][2][3][4] . Advances in rapid genomic sequencing has enabled the development of personalized cancer vaccines (PCV) targeting tumor-specific mutations termed neoantigens (neoAg). Early studies in mice 5,6 and phase I clinical trials 7-10 demonstrate the feasibility of PCV to generate neoAg T cell responses; however, the magnitude of CD8 + T cell responses has been limited to date 10 .
An important variable in improving CD8 + T cells responses with PCV is the vaccine platform. Accordingly, we developed a self-assembling nanoparticle vaccine platform to standardize the delivery of long peptides (LP) 11 . At 20-50 nm in size, the nanoparticles allow for efficient drainage via lymphatics and uptake by dendritic cells (DC) that prime CD8 + T cells 12 . Further, co-delivery of the antigen and TLR7/8 agonist (TLR7/8a) ensures DC maturation via TLR activation in the same cell that has acquired antigen, to achieve optimal presentation to CD8 + T cells. Indeed, the SNP-7/8a vaccine platform generated higher magnitude and breadth of CD8 + T cells with antitumor efficacy when benchmarked against the most commonly used PCV approaches 11 .
In addition to magnitude, the quality of CD8 + T cell responses can be an important determinant in protection against viral infections and tumors 13,14 . Studies elucidating the transcriptional and epigenetic heterogeneity of CD8 + T cells has provided important advances in understanding lineage differentiation, proliferative potential and functional capacity. In chronic lymphocytic choriomeningitis virus (LCMV) infection, TCF1 was identified as a critical transcription factor that maintains a precursor population within the exhausted T cell (Tex) pool [15][16][17][18][19] . Highly expressed in naive CD8 + T cells, TCF1 is downregulated during effector cell differentiation in response to pro-inflammatory cytokines such as IL-12 and type I interferons (IFN-I) 20,21 . Previously thought to be dysfunctional, a subset of PD-1 + Tex referred to as progenitor exhausted or stem-like cells that express TCF1 remain responsive to checkpoint inhibitors (CPI); they maintain stem cell-like quality by retaining the capacity to proliferate and self-renew [22][23][24] . Studies assessing CD8 + tumor infiltrating lymphocytes (TIL) in humans identified gene signatures of stem-like cells including Tcf7 (encoding TCF1), associated with better prognosis and response to checkpoint blockade [25][26][27][28][29][30] . Adoptive transfer studies in mice showed that the self-renewing capacity of TIL was regulated by TCF1 31 whereas cells lacking TCF1 were less effective in controlling tumor growth 32 . Collectively, these data suggest an important role for TCF1 + PD-1 + CD8 + T cells in mediating protection against viral infections and tumors.
Here, we demonstrate how altering the route and dose of vaccination with SNP-7/8a influences the magnitude, transcriptional quality and antitumor capacity of neoAg-specific CD8 + T cells. In addition, we identify the innate mechanisms for how the route of vaccination alters antigen duration and tropism for distinct DC subsets critical for imprinting CD8 + T cell responses.

RESULTS
Route and dose of SNP-7/8a immunization alter the magnitude and quality of neoAg + CD8 + T cells SNP-7/8a is a self-assembling nanoparticle vaccine platform that standardizes the delivery of LP 11 (Fig. 1a). Here, we determined how modifying the dose and route of SNP-7/8a immunization influenced CD8 + T cell responses. Mice were vaccinated with SNP-7/8a containing Reps1, an MC38 murine colon carcinoma neoAg (Fig. 1b). Whole blood was collected to measure neoAg-specific CD8 + T cells by tetramer staining (Fig. 1c and Extended Data Fig. 1a) or IFN-γ after re-stimulation with Reps1 peptide (Extended Data Fig. 1b). At a dose of 8 nmol, subcutaneous administration of SNP-7/8a (SNP-SC) generated 20-fold higher neoAg-specific CD8 + T cells compared to IV vaccination (SNP-IV) (Fig. 1d). CD4 + T cells produced IFN-γ at low frequencies as previously described 11 (Extended Data Fig. 1c). Together, these data show that the route of vaccination alters the magnitude of the CD8 + T cell response.
Next, we characterized CD8 + memory precursor effector cells (MPEC) or short-lived effector cells (SLEC) based on differential expression of CD127 (IL-7R) and KLRG1 (Fig.  1e). A high proportion of neoAg-specific CD8 + T cells following SNP-SC were SLEC (~60% of tetramer + ) whereas SNP-IV cells were primarily MPEC (~60% of tetramer + ) ( Fig.  1f and Extended Data Fig. 1d). The frequency of MPEC is inversely correlated to the frequency of tetramer + CD8 + T cells ( Fig. 1g and Extended Data Fig. 1e). We then assessed expression of PD-1 and Tim-3, canonical markers of T cell activation, exhaustion or severe dysfunctionality 33 (Fig. 1h). Tetramer + CD8 + T cells from both SNP-SC and SNP-IV mice were PD-1 + but only SC-vaccinated groups expressed low levels of Tim-3 ( Fig. 1i and Extended Data Fig. 1f). Peptide re-stimulation markedly increased the expression of PD-1 and Tim-3 following SNP-SC but not SNP-IV ( Fig. 1j and Extended Data Fig. 1g). Taken together, the data suggest that the route of vaccination influences the differentiation of neoAg-specific CD8 + T cells.

IV administration of SNP-7/8a generates TCF1 + PD-1 + CD8 + T cells with antitumor capacity upon anti-PD-L1 treatment
To investigate the antitumor capacity of neoAg-specific CD8 + T cells, mice were challenged with MC38 tumors (Fig. 2a). To evaluate the effect of a checkpoint inhibitor (CPI), mice were also either treated with or without anti-PD-L1. Anti-PD-L1 treatment alone was not sufficient to control tumor growth in unvaccinated mice ( Fig. 2b and Extended Data Fig. 2a) or extend survival (Fig. 2c). SNP-SC vaccinated mice significantly controlled tumor growth compared to naive mice (Fig. 2b) independent of CPI treatment. Despite the 10-fold reduction in CD8 + T cell responses (Fig. 2d), SNP-IV vaccinated mice were able to control tumor growth (Fig. 2b) and extend survival compared to naive (Fig. 2c) only when combined with CPI treatment.
The distribution of tetramer + CD8 + T cells was assessed at the time of tumor challenge in blood, spleen, popliteal LN (SC vaccine-draining) and the lungs (Fig. 2e). NeoAg-specific CD8 + T cells generated by SNP-SC was significantly higher in all tissues compared to SNP-IV. We detected a population of TCF1 + PD-1 + within tetramer + cells in the spleen (Fig. 2f). Such cells have been described in chronic LCMV 15 as a population of exhausted cells that retain proliferative capacity. SNP-IV generated a higher proportion of TCF1 + cells, representing ~50% of tetramer + CD8 + T cells (Fig. 2g). TCF1 + cells in the SNP-IV group exhibited high CD127 and low KLRG1 (Fig. 2h). TCF1 + CD8 + T cells generated by SNP-IV also expressed proteins upregulated by Tex progenitor cells that retain a stem-like program 15,18,27 such as eomesodermin (EOMES) and CXCR3 (Fig. 2h). However, CXCR5 expressed by stem-like cells following chronic LCMV was not detected in TCF1 + CD8 + T cells following SNP-7/8a vaccination. As a staining control, CD4 + T follicular helper (Tfh) cells expressed CXCR5 (Extended Data Fig. 2b). In contrast, TCF1 + CD8 + T cells generated by SNP-SC had high expression of KLRG1 and lower EOMES levels (Fig. 2h). To assess the generalizability of these observations, mice were immunized with SNP-7/8a containing other antigens including E7, OVA and Trp1 (Extended Data Fig. 2c). Similarly, SNP-IV resulted in lower magnitude responses (Extended Data Fig. 2d), but a higher frequency of TCF1 + PD-1 + cells (Extended Data Fig. 2e and 2f) and MPEC (Extended Data Fig. 2g and 2h) compared to SNP-SC, regardless of the antigen. These data indicate a phenotypic difference in the neoAg-specific CD8 + T cells generated depending on route of vaccination.

Single cell analysis of neoAg-specific CD8 + T cells identifies stem-like gene signature following SNP-IV
To further characterize the neoAg-specific CD8 + T cells, we sorted tetramer + cells following SNP-SC and SNP-IV vaccination ( Fig. 3a and Extended Data Fig. 3a). Tetramer + CD8 + T cells from individual animals were barcoded prior to single-cell RNA sequencing (scRNAseq) using the droplet-based system of 10x Genomics (Extended Data Fig. 3b). Cells were clustered based on gene expression using an unsupervised inference analysis (Monocle 3). The 12 clusters identified were visualized by uniform manifold approximation and projection for dimension reduction (UMAP) algorithm (Fig. 3b). Clustering data showed separation of single cells by route of vaccination ( Fig. 3c and Extended Data Fig. 3c). SNP-IV neoAg + cells were primarily in clusters 2 and 4, which we refer to as "stem-like" cells given that Tcf7 (encoding TCF1) was a significant differentially expressed gene (DEG) in these two clusters ( Fig. 3d and Supplementary Table 1). In contrast, SNP-SC neoAg + cells mainly appeared in the "effector" cluster consisting of clusters 1, 3, 5, 7 and 8 (Fig. 3d). Both stem-like and effector clusters form stable states (high density areas on UMAP) 34 (Extended Data Fig. 3d). Cluster 10 represents a small population of naive T cells (Ccr7, Lef1, Sell, Dapl1, Igfbp4) (Extended Data Fig. 3e) that may have contaminated while sorting due to low frequencies with SNP-IV. Excluding naive cells, the T cell receptor (TCR) repertoire is largely diverse following both routes of immunization (Extended Data Fig. 3f). The fraction of stem-like or effector cells within each clone is driven by the route of vaccination, rather than the clonotype identity (Extended Data Fig. 3g).
Next, we reconstructed a developmental trajectory of neoAg-specific CD8 + T cells. The pseudotime analysis showed a trajectory originating from naive T cells, branching into stemlike cells and eventually effector cells (Fig. 3e). We further compared stem-like cells in this study with published datasets from chronic LCMV or tumor models 18,22,32 , identifying 337 shared genes and 50 unique genes in our dataset ( Fig. 3f and Supplementary Table 2). Applying a list of previously described stem-like genes (also referred to as 'Tex progenitor') and effector genes ('Teff') in the context of LCMV infection 18 , we confirmed that the stemlike gene signature was highly expressed in clusters 2 and 4, whereas the effector gene signature was more pronounced in clusters 1, 3, 5, 7 and 8 ( Fig. 3g).
Consistent with flow cytometry detection of TCF1 in SNP-IV CD8 + T cells (Fig. 2f), the stem-like cluster expressed high levels of Tcf7 (Fig. 3g). These cells also expressed genes associated with T cell exhaustion: Tox, Lag3, and Ctla4, yet upregulated anti-apoptotic genes such as Bcl2 (Supplementary Table 2). As previously observed in stem-like CD8 + T cells from chronic LCMV 18,19,35 , Slamf6 (encoding Ly108) was highly expressed in the stem-like CD8 + T cells following IV vaccination. Unlike data from LCMV studies, the stemlike CD8 + T cells reported here did not show increased Cxcr5, consistent with the flow cytometric data (Supplementary Table 2 and Extended Data Fig. 3h). The stem-like cluster expressed high levels of Xcl1, which encodes for XCL1 -the ligand for XCR1 + cDC1. The effector cluster expressed genes encoding cytotoxic granules such as Gzma and Gzmb as well as inhibitory receptors such as Klrg1 ( Fig. 3h 19,35 , is also highly expressed in our effector cluster. Other notable genes that are highly expressed in the effector cluster include Zeb2 (triggers cytotoxic T lymphocytes to adopt terminally differentiated state 36 ), Lgals1 (Galectin-1, a pro-apoptotic molecule 37 ) and S1pr5 (S1PR5, cues exit from lymphoid tissue 38 ) ( Fig. 3g and 3h).
To further evaluate cellular states based on expression of transcription factors and target genes, we measured regulons' activity by performing SCENIC analysis 34 (Fig. 3i). Changes in transcriptional network can be detected along the Monocle 3 pseudotime trajectory. Upregulation of Eomes regulon (Eomes and 21 related genes) can be measured in the stemlike cluster, in line with EOMES detected by flow cytometry (Fig. 2h). In contrast, the effector cluster is marked by upregulation of several regulons including Runx3, Runx2, Rora and Fli1 (Fig. 3i). Taken together, at the transcriptional level, the neoAg-specific CD8 + T cells generated by SNP-IV express a stem-like gene signature, whereas SNP-SC cells express an effector gene signature. Further, trajectory inference analysis of scRNA-seq data highlights differences in the developmental states of stem-like and effector CD8 + T cells.

Therapeutic vaccination by SNP-IV controls tumor growth but not SNP-SC
To assess the antitumor capacity of SNP-7/8a in mice with established tumors, MC38 was implanted and vaccination was administered on day 7 (prime) and day 14 (boost) (Fig. 4a). Anti-PD-L1 was given at the time of boost. In this study, the dose of SNP-IV was increased to 32 nmol to generate CD8 + T cells of comparable magnitude to the SNP-SC vaccinated mice (Fig. 4b). Both vaccination routes resulted in ~10% Reps1 + CD8 + T cells (Fig. 4c). Despite similar numbers of neoAg-specific CD8 + T cells, SNP-SC mice could not control tumor growth whereas SNP-IV mice significantly controlled tumor growth compared to naive mice (Fig. 4d). The antitumor efficacy of SNP-IV is antigen-specific and adjuvantdependent as vaccinating with an irrelevant antigen IV (Fig. 4e) or no adjuvant (SNP with no TLR7/8a) (Extended Data Fig. 4a) was ineffective. The differences in tumor growth is further confirmed in prolonged survival of mice vaccinated with SNP-IV but not SNP-SC (Fig. 4f). Decreasing the SNP-SC dose or reducing to a single vaccination was not effective (Extended Data Fig. 4b). The tumor control (Fig. 4g) and increased survival ( Fig. 4h) by SNP-IV were abrogated by depleting CD8 + T cells but not CD4 + T cells or NK cells (Extended Data Fig. 4c and 4d).
To delineate each variable of the SNP-IV treatment regimen, MC38 tumor-bearing mice were vaccinated on day 7 with 32 nmol of SNP containing Reps1 (Fig. 4i). On day 14, groups of mice were either left untreated ("prime"), given CPI ("prime and CPI"), vaccinated again ("prime and boost"), or vaccinated again together with CPI ("prime, boost and CPI"). Indeed, the combination of both vaccinations and CPI was most effective at controlling tumor growth ( Fig. 4i) and extending survival (Fig. 4j). Spleens were collected from mice on day 14 (post prime) and day 21 post tumor implantation (Fig. 4k). Without the boost or CPI treatment, the frequency of neoAg-specific CD8 + T cells in the spleen was significantly lower (6-fold) than mice that received a boost and CPI (Fig. 4l). A clear expansion of neoAg-specific CD8 + T cells was observed when enumerating tetramer + CD8 + T cells in the spleen on day 21 (Fig. 4m). On day 14, approximately 80% of CD8 + T cells were stem-like (TCF1 + Granzyme B − ) after SNP-IV, compared to 35% following SNP-SC (Fig. 4n). Co-staining with PD-1 confirmed that these cells were high in PD-1 (Extended Data Fig. 4e). By day 21 post SNP-IV, neoAg + CD8 + T cells from spleens of tumor bearing mice have mostly differentiated to effector cells (TCF1 − Granzyme B + ) (Fig. 4n). Mice receiving both immunizations and CPI maintained high numbers of stem-like cells in the spleen ( Fig. 4o), unlike the other groups that did not receive a boost or CPI. This coincided with a significant 10-fold increase in neoAg + effector cells expressing Granzyme B in mice that received boost and CPI, compared to mice that received boost with no CPI (Fig. 4p). In contrast, a modest 1.5-fold expansion of effector cells in the spleen was observed following SNP-SC (Extended Data Fig. 4f). Importantly, neoAg-specific CD8 + T cells generated by SNP-SC co-expressed PD-1 and Tim-3 by day 21 (~35%) while SNP-IV cells did not express Tim-3 in the spleen (Extended Data Fig. 4g). On day 21, SNP-IV cells had increased Ki-67 expression in blood and spleen (Extended Data Fig. 4h), indicating an expansion of circulating effector cells. Taken together, these highlight the capacity of stem-like neoAg + CD8 + T cells generated by SNP-IV to proliferate and replenish effector cells for antitumor responses upon CPI treatment.

Prolonged antigen persistence, DC uptake and activation following SNP-SC but not SNP-IV
To investigate differences in the mechanism of CD8 + T cell priming, fluorescently labeled SNP-7/8a was used to visualize the distribution and uptake by DC. In vivo imaging of mice revealed that SNP-SC remain localized to the site of vaccination two weeks post vaccination, highlighting a depot effect (Extended Data Fig. 5a). In contrast, SNP-IV resulted in systemic distribution of the vaccine detectable up to 24 hours by in vivo imaging (Extended Data Fig.   5a). LN sections analyzed by confocal microscopy confirmed that vaccine can be detected up to two weeks after SNP-SC, primarily in the T cell zones. (Fig. 5a and Extended Data Fig. 5b). Co-staining with monocyte, macrophage and DC markers revealed co-localization of vaccine with cells expressing CD11b and CD11c (Extended Data Fig. 5c). Following SNP-IV, confocal images of spleen sections show vaccine uptake by CD11b + CD11c + cells situated in the marginal zones primarily after 6 and 24 hours with minimal vaccine detection after 3 and 7 days (Fig. 5b). Popliteal LN and spleens were harvested for flow cytometric analysis (Extended Data Fig. 5d). Labeled vaccine could be detected in both cDC1 (Lin − MHCII + CD11c + XCR1 + ) and moDC (Lin − CD64 + F4/80 + MHCII + CD11c + ) in the popliteal LN after SNP-SC as late as 3 weeks after vaccination ( Fig. 5c and Extended Data Fig. 5e). Similarly, SNP-IV resulted in uptake by cDC1 and moDC in the spleen. However, this peaked at 6 hours post vaccination ( Fig. 5c and Extended Data Fig. 5f). Co-stimulatory molecules CD80 and CD86 as well as the regulatory marker PD-L1 were upregulated in cDC1 and moDC after vaccination by both routes (Fig. 5d). SNP-IV resulted in transient DC activation that peaked at day 1, whereas SNP-SC resulted in prolonged activation of DCs over three days (Fig. 5d). Expressions of CD80, CD86 and the migratory marker CCR7 on moDC were detectable 3 weeks later suggesting continued infiltration of APC into the draining LN (Extended Data Fig. 5g). The frequency of cDC1 in the spleen after SNP-IV peaked at 6 hours and diminished to below baseline levels at 24 hours as assessed by flow cytometry (Extended Data Fig. 5h and 5i). In contrast, the numbers of cDC1 remain high three weeks after SNP-SC in the popliteal LN (Extended Data Fig. 5h and 5i). The differences in presence and activation of cDC1 at the site of priming highlight the potential importance of DC in modulating CD8 + T cell quality after SNP vaccination.

cDC1 and moDC are required for CD8 + T cell priming
To directly address the role of specific APCs in priming CD8 + T cells, wild type (WT), Batf3 −/− and Ccr2 −/− mice lacking cross-presenting cDC1 and circulating monocytes respectively were immunized with SNP-7/8a (Extended Data Fig. 6a and 6b). The frequency of neoAg-specific CD8 + T cells was significantly decreased following SNP-SC in Batf3 −/− (4-fold lower) and Ccr2 −/− (20-fold lower) mice (Fig. 6a). As monocytes remain in the spleens of Ccr2 −/− mice, we generated bone marrow chimeras of Ccr2 DTR mice to conditionally deplete monocytes in peripheral tissues and lymphoid organs (Extended Data Fig. 6c and 6d). Following SNP-IV, the frequency of neoAg-specific CD8 + T cells significantly decreased by diphtheria toxin (DT) treatment of CCR2 DTR BM chimera mice (5-fold lower) and Batf3 −/− mice (15-fold lower) suggesting dependency on monocytes and cDC1 (Fig. 6b). Mice receiving WT BM with or without DT treatment and mice receiving Ccr2 DTR BM without DT treatment generated similar frequencies of neoAg-specific CD8 + T cells after SNP-IV (Extended Data Fig. 6e). Collectively, these data highlight a critical role for not only Batf3-dependent cDC1 but also moDC in priming CD8 + T cells with the SNP-7/8a vaccine.
As type I IFN (promoting cross-presentation 39 ) and IL-12 (promoting Th1 skewing 40 ) can have critical roles in controlling CD8 + T cell priming, these cytokines were measured. SNP-IV induced transient production of IL-12 and IFN-α which peaked at 6 hours post vaccination in blood and spleen ( Fig. 6c and Extended Data Fig. 6f). In contrast, IL-12 was detected up to two weeks after SNP-SC in the popliteal LN (Fig. 6c) consistent with the duration of vaccine detection (Fig. 5a).

SNP-SC leads to prolonged antigen retention and presentation in vivo
Based on the data showing prolonged retention of vaccine and innate activation following SC immunization ( Fig. 5c and 5d), we hypothesized that antigen persistence mediated the differences in CD8 + cell quality between SC and IV vaccination. Naive CD45.2 OT-I CD8 + T cells were labeled with a fluorescent dye and transferred to CD45.1 congenic mice 1, 3 or 7 days after vaccination with SNP-7/8a delivering the OT-I peptide (SIINFEKL) (Fig. 6i). Spleens were collected 4 days after cell transfer to assess the extent of CD8 + T cell proliferation as an indicator of antigen presence. At all three time points, cell proliferation could be detected, with the largest numbers of OT-I measured being 7 days after SNP-SC vaccination (Fig. 6j). In contrast, SNP-IV resulted in a burst of OT-I expansion when transferred 1 day after vaccination followed by significantly lower cell numbers 3 and 7 days after vaccination (Fig. 6j). Overall, the prolonged persistence of antigen in the popliteal LNs drove T cell differentiation, thus explaining the high magnitude but lower proliferative potential of neoAg-specific CD8 + T cells generated by SNP-SC.

DISCUSSION
In this study, we report how SNP-7/8a dose and route control the magnitude and transcriptional quality of neoAg-specific CD8 + T cells. In the context of cancer immunotherapy, persistence and functional capability are important factors given the chronic nature of cancer. The most striking finding was the demonstration that IV vaccination led to a major difference in the phenotypic and transcriptional quality of the CD8 + T cell response compared to SC vaccination. SNP-7/8a vaccination enables specific generation of stem-like or effector cells by modifying the route of vaccination. We demonstrate how this can be achieved in vivo using CD8 + T cells primed from a polyclonal naive repertoire, without the caveats of TCR-transgenic systems using model antigens. We would note that IV administration of RNA-based PCV has been shown to be more immunogenic than direct LN injection; however the transcriptional quality of CD8 + T cells by this route has not been assessed 41,42 . Here we clearly demonstrate that neoAg-specific CD8 + T cells generated by SNP-IV have a high proportion of TCF1 + PD-1 + stem-like cells and mediate antitumor effects dependent on CPI.
The scRNA-seq data of neoAg + CD8 + T cells revealed distinct transcriptional landscapes following SNP-SC or SNP-IV, providing insight into the dynamics of CD8 + T cell activation and differentiation following vaccination: naive cells give rise to stem-like cells that can be further differentiated into effector cells. Comparison of clusters revealed several novel genes highly expressed by stem-like CD8 + T cells. Xcl1 expression by stem-like CD8 + T cells suggests a role for XCR1 + cDC1 43 in licensing their activity in the spleen. In stem-like cells, the expression of Tox, recently described as a crucial regulator of T cell exhaustion 44,45 , may be beneficial in preventing overstimulation of CD8 + T cells leading to cell death especially in the context of cancer where there is antigen persistence. Interestingly, neoAgspecific CD8 + T cells generated by SNP-IV resemble the stem-like or progenitor exhausted cells described in chronic LCMV infection, whereas cells generated by SNP-SC share genes upregulated in acute LCMV infection 15,18,19 . Epigenetic analysis, specifically around the Tox locus, may reveal whether vaccine-induced CD8 + T cells resemble a distinct developmental program or share specific features of exhausted cells identified in LCMV 46,47 . Unlike chronic LCMV where there is persistent antigenic stimulation, we observe stem-like CD8 + T cells following IV vaccination of a peptide-based vaccine where the duration of antigenic delivery peaked at six hours. These data suggest distinct mechanisms of CD8 + T cell priming in vaccination versus natural infection and demonstrate the rapidity by which such cells can be induced by the SNP-7/8a vaccine which will be critical for therapeutic tumor vaccination.
The covalent linking of antigen and adjuvant into a nanoparticle was designed to synchronously deliver the innate stimulation with the antigen for efficient cross-presentation. Several important factors may contribute to the modulation of CD8 + T cell quality upon IV vaccination: location of CD8 + T cell priming, actual dose of vaccine delivered to splenic DC and degree of inflammation. The duration of antigen persistence of up to 3 weeks in the draining LN by SNP-SC contrasted with a short burst of antigen in the spleen by SNP-IV. Despite the brief duration, the spleen remains a large reservoir for DC as noted by the tenfold increase in absolute numbers of cDC1 or moDC acquiring vaccine at the peak of SNP-IV delivery, compared to numbers of vaccine + DC in the popliteal LN at the peak of SNP-SC delivery. This is likely the key factor in regulating the magnitude and quality of responses respectively. In a different cancer vaccine model using peptide and incomplete Freund's adjuvant, antigen persistence also led to dysfunctional CD8 + T cells 48 . However, it is notable in the prophylactic tumor model presented here, the CD8 + T cells generated by SC vaccination were still highly functional. Confocal images of LN sections showed that, while antigen was retained for 3 weeks in the LN after SC vaccination, the localization of antigen changed: in the first 24 hours, antigen was mostly in the subcapsular sinus, but over the next two weeks, antigen concentrated into the T cell zones. This slow-release effect has been the basis of other rational vaccine design, regulating the kinetics of antigen exposure 49 . Thus, SNP-SC was efficient at generating effector CD8 + T cells at high numbers. This approach may be valuable for prophylactic cancer vaccines targeting known shared tumor antigens.
Importantly, co-delivery of antigen and adjuvant permits systemic IV administration by limiting the tolerogenic effects of having free peptide taken up by APC in the absence of a danger signal. The short-lived pharmacokinetics of SNP-IV in the spleen likely accounts for the lower magnitude when given at the same dose as SNP-SC. The reduction in numbers of splenic cDC1 between 24 hours and 3 days after IV vaccination may serve as a negative immunoregulatory mechanism. Further, the dependency on moDC for CD8 + T cell responses suggests that these cells either contribute indirectly by providing the required cytokines for T cell activation, by transferring antigen to other DC, or directly by presenting antigens to CD8 + T cells. Following SNP-IV, the frequency of cDC1, the canonical subset for efficient cross-priming, were dramatically reduced. We hypothesize that moDC, generally considered less efficient APC compared to cDC1 50 , may be responsible for providing less-differentiated, more stem-like CD8 + T cells. Future studies will focus on how these distinct DC subsets can modify both the magnitude and quality of CD8 + T cells in the context of vaccination.
In conclusion, this study shows that the route of administration of SNP-7/8a vaccine substantially affected the magnitude and transcriptional quality of neoAg specific CD8 + T cell responses, which had a corresponding impact on functionality and therapeutic outcomes. These findings have implications in the clinical development of therapeutic vaccines for cancer patients.

Mice
Female B6 mice (C57Bl/6J), Ccr2 −/− , Batf3 −/− , Ifnar −/− , Tlr7 −/− , Il12b −/− were purchased from Jackson laboratories and maintained in pathogen-free conditions. OT-I Rag2 transgenic mice were purchased from Taconic. CCR2 DTR mice 51 were bred inhouse. Mice were used in studies when 6-8 weeks old. All animal experiments were performed at the Vaccine Research Center (VRC) at the National Institutes of Health (NIH, USA) with the approval of the Institutional Animal Care and Use Committee (ACUC) at the NIH. Experiments complied with the ethical guidelines set by ACUC and animals were humanely euthanized at defined endpoints.

Generation of bone marrow chimeras
8-week-old recipient mice received 13 Gy of γ-irradiation (2 doses of 6.5 Gy each) prior to IV reconstitution with bone marrow from CCR2 DTR mice. Four to eight weeks after reconstitution, successful chimerism was assessed by flow cytometry. Mice were used in studies eight weeks after reconstitution. Baharom

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Vaccines
SNP conjugate vaccines were produced as previously described 11 . Briefly, peptide antigens (Genscript, USA) were linked to hydrophobic blocks containing Imidazoquinoline-based TLR7/8a (Avidea Technologies, USA) using a click chemistry reaction. For pharmacokinetics studies, SNP conjugate vaccines were produced by linking Alexa Fluor 647 to hydrophobic blocks.

Cells
MC38 cell line was a kind gift from Lélia Delamarre (Genentech, USA). Working cell banks were generated immediately upon receipt and used for tumor experiments. Cells were tested regularly for mycoplasma contamination, and none tested positive throughout the studies.

Tumor implantation
MC38 tumor cells were cultured in complete Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal calf serum (Atlanta Biologics), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco), 1x non-essential amino acids (GE Life Sciences) and 1 mM sodium pyruvate (GE Life Sciences). For each tumor implantation, a frozen cell aliquot was thawed and cultured in media at 37°C and 5% CO 2 , passaged once, and harvested using trypsin EDTA (Gibco). 10 5 cells in sterile PBS per mouse were implanted subcutaneously on the right flank. Tumors were measured twice a week using digital calipers. Tumor volume was estimated using the formula: [tumor volume = short × short × long / 2]. Animals were euthanized when tumors reached size criteria of 1000 mm 3 .

Blood and tissue processing
Heparin-treated blood was collected and lysed with ACK lysis buffer (Quality Biologicals). Lungs, liver, kidneys and tumors were harvested in digestion media containing RPMI-1640, 10% fetal calf serum, 50 U/mL DNAse I (Sigma) and 0.2 mg/mL collagenase D (Sigma). Tissues were mechanically disrupted using the respective programs on the gentleMACS dissociator (Miltenyi Biotech, Germany) and incubated at 37°C for 30-45 min in a shaking incubator. Spleens were mechanically disrupted and lysed with ACK lysis buffer. Lymph nodes were mechanically disrupted in BioMasher tubes (Nippi Inc, Japan). All single cell suspensions were filtered through a 40 μm nylon mesh filter plate (EMD Millipore) or 70 μm cell strainer and resuspended in PBS for flow cytometry staining. For ex vivo cultures, cells were resuspended in RPMI-1640 media (GE Life Sceiences) supplemented with 10% heatinactivated fetal calf serum (Atlanta Biologics), L-Glutamine-Penicillin-Streptomycin (Sigma Aldrich), non-essential amino acids (GE Life Sciences), sodium pyruvate (Hyclone), HEPES buffer (Hyclone) and beta-mercaptoethanol (Sigma).

Flow cytometry
For T cell tetramer analysis, cells were assessed for viability with LIVE/DEAD Fixable Blue Cells were acquired on an LSRFortessa X50 (BD) using FACSDiva software (BD) and analyzed on FlowJo software (BD).

OT-I in vivo proliferation assay
Spleens and lymph nodes were harvested from CD45.2 OT-I mice and labeled with Tag-it Violet Proliferation and Cell Tracking Dye (Biolegend) as per manufacturer's protocol. Labeled cells (2×10 6 ) were transferred via retro-orbital intravenous injection to each congenic CD45.1 mouse at different time points after vaccination. Four days after transfer, spleens were harvested from recipient mice for flow cytometric analysis.

In vivo imaging
Whole body imaging of mice following immunization with Alexa Fluor 647-labeled vaccines was performed using IVIS Spectrum (Perkin Elmer).

Cell sorting for scRNA-seq
Spleens from mice vaccinated with SNP via SC or IV injections were collected 2 weeks post boost and processed into a single cell suspension by mechanical dissociation. Splenocytes were stained with Reps1 tetramer and hashtag antibodies (Biolegend Total-Seq-C antibodies 1-5) 52 . CD8 + T were isolated by fluorescence activated cell sorting (FACS) into 1.5 mL Eppendorf tubes containing staining buffer (2% FBS/PBS). Up to 4×10 4 cells were sorted per mouse. Cells from mice in both treatment groups with distinct hashtags were mixed to form two pools with an aim for equivalent numbers of cells from each mouse. Each pool of cells was loaded in duplicate in a Chromium single cell sorting system (10X Genomics). Expression and hashtag library construction was performed following Chromium Single Cell VDJ Library protocol with a loading target of 1 ×10 4 cells per lane. The resulting 4 libraries were pooled prior to sequencing on a Novaseq 6000 S1 chip.

Data processing for scRNA-seq
Raw sequencing files were aligned to the mouse mm10 genome using Cell Ranger software (10X Genomics). Hashtag data was added to each expression library using Seurat V3.1 and demultiplexed using the HTODemux function. After demultiplexing, singlet cells identified by hashtag from each of the libraries were pooled into a single Seurat object. Cells were further filtered by excluding those with greater than 5% mitochondrial genes in their library and fewer than 1000 genes 18 . 19,368 cells remained after filtering and were used for downstream analysis.

Downstream analysis of scRNA-seq data
Principal component analysis (PCA), uniform manifold approximation and projection (UMAP) for dimension reduction 53 , clustering the cells (by Leiden method), constructing trajectory and pseudotime analyses 54 were performed on UMI counts using Monocle 3 (R package, version 0.2.1) 55 . Analysis of differentially expressed genes (DEG) and heatmaps were performed on the Log normalized data with Seurat (R package, version 3.1.4) 56 . The differential expression among the clusters was tested by the non-parametric Wilcoxon rank sum test, followed by Bonferroni correction method (using all features) to calculate adjusted P value. Top DEG were visualized using SeqGeq software (BD). SCENIC pipeline (R package, version 1.1.2.2) was used to construct and score gene regulatory network (regulons) as described previously. 34 Each regulon is composed of a transcription factor and its putative target genes. The output of SCENIC is a matrix of regulons' activity, in which rows correspond to regulons and columns correspond to cells.

TCR analysis of scRNA-seq data
T cell receptor (TCR) libraries were generated independently for all four 10X lanes according to the Chromium Single Cell VDJ library protocol. Raw sequencing files were aligned to the mouse mm10 genome and annotated with their TCR genes by using the cellranger vdj function from the Cell Ranger software suite (10X Genomics). We excluded cells that did not have full length or productive TCR alpha (TRA) or beta (TRB) gene pairs and those with multiple gene pairs (duplicates). Filtered TRA/TRB gene pairs were matched to the cells used for gene expression analysis by 10X barcode and any TRA/TRB pair that was not present in our gene expression data set was removed from further analysis. Clonotype was assigned based on the complementarity-determining region 3 (CDR3) amino acid sequence of the TRA/TRB gene pair. Only clonotypes expressed by more than 100 cells were visualized in the heat map.

Enzyme-linked immunosorbent assay
Serum from whole blood, spleens and popliteal lymph nodes were collected at specified time points after vaccination. Supernatants were collected from single cell suspensions of spleens and LNs that were cultured in vitro for 12 hours in complete RPMI at 37°C. Commercially available ELISA kits were used to measure IL-12p40 (Peprotech) and all subtypes of IFN-α (PBL Assay Science) as per manufacturer's protocols.

Statistical analysis
All results are presented as median with standard deviation. Statistics were assessed by Kruskal-Wallis with Dunn's correction for multiple comparisons, two-way ANOVA with Bonferroni correction, Log-rank test and Mann Whitney test for immunogenicity (Graphpad Prism).

Data availability
The data that support the findings of this study are available from the corresponding author upon request. The scRNA-seq data have been uploaded to Gene Expression Omnibus (accession number: GSE158240).

Extended Data
Extended Data Fig. 1 |. Route and dose of SNP-7/8a immunization controls the magnitude and phenotype of antigen-specific CD8 T cells. a, Whole blood was collected on day 21 to measure the frequency of tetramer + CD8 T cells post boost. Bar graphs summarize the frequency of tetramer + CD8 T cells from blood (n = 10). b, Bar graphs summarize the frequency of IFNγ + CD8 T cells from blood (n = 10) on day 21. c, Bar graphs summarize the frequency of IFNγ + CD4 T cells from blood (n = 10).

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.