Introduction

Cancer immune checkpoint inhibition (ICI) therapy involves the intravenous (i.v.) infusion of biologics to suppress co-inhibitory T-cell signaling in order to prime and sustain tumor-specific, cytotoxic T-cells in the expanse of the tumor microenvironment (TME). Yet, immunology teaches us that systemic immunity is established regionally within the confines of regional draining lymph nodes (dLNs), where the assembly of antigen (Ag), Ag presenting cells, cytokines, chemokines, as well as naïve T- and B- cells, orchestrate robust Ag-specific, activated T-cell production. 

In this work, we sought to show that regionally dosing of ICI and vaccine to non-tdLNs can create tumor specific T-cell priming and maximize anti-tumor immune responses. 

Methods

We compared tumor responses to  αCTLA-4 (5 mg/kg) delivered i.v. and intradermally (i.d.) to non-tumor dLNs (non-tdLNs) contralateral to B16F10 implant in the mouse hind limb. Given that B16F10 is an immunologically “cold” tumor that is non-responsive to systemic ICI monotherapies, we sought to enhance responses through ICI delivery to non-tdLNs that also received virus-like particle (VLP), Qβ(1668) vaccination and boost against tumor neoantigen PMEL17. Animals were vaccinated on days 7 and 14 following implant and αCTLA-4 was dosed on days 7, 9, and 11 with sacrifice and tumor harvest a week later.

Flow cytometry, scRNA-seq, and single cell T-cell receptor (TCR) sequencing was performed on harvested tumors after CD45+ enrichment. We analyzed the proportional changes and TCR clonal expansion in CD45+CD3+ tumor infiltrating lymphocyte (TIL) subpopulations from control animals, animals dosed with αCTLA-4, i.v., and αCTLA-4 i.d. + Qβ(1668)-PMEL17. 

Results

When compared to αCTLA-4 i.v., vaccination combined with i.d. dosing of αCTLA-4 to non-tdLNs resulted in significant anti-tumor responses as measured by tumor volume.  Systemic dosing caused 5-6 fold fewer CD4⁺Foxp3^- cells, fewer CD8+ effector cells expressing cytokines, and 6-7 fold more CD8⁺ TILs with an exhausted phenotype compared to i.d. dosing of αCTLA-4  in non-tdLNs receiving vaccine. TILs from animals receiving αCTLA-4 i.d. and Qβ(1668)-PMEL17 exhibited more CD4⁺Foxp3^- consistent with induced priming of CD4+ T-cells.

Surprisingly, we found expansion of γδ T-cells represented in the TME of animals dosed regionally with αCTLA-4 i.d., and Qβ(1668)-PMEL17 while γδ T-cells were obliterated in animals dosed systemically.

We found 768 clonotypes of CDR3 sequence in animals dosed regionally with VLP and αCTLA-4. In comparison, TILs from untreated littermates and littermates dosed systemically with αCTLA-4 expressed far fewer 190 and 409 clonotypes, respectively. We also identified potential clonotypes highly specific to PMEL17 as determined from comparison of TCR sequence (GSM5494125) using CDRdist similarity measures.

The top CD8+ clonotypes were shared between cytolytic and exhausted T-cell populations in animals dosed systemically while the animals dosed with VLP and αCTLA-4 to the non-tdLNs had top CD8+ clonotypes specific to PMEL17 expanded in different cytolytic populations. Functional analysis shows that the cytolytic TILs in systemically dosed animals were most enriched in INFβ responses that modulate cytokine expression and inhibit T-cell activation and proliferation while the cytolytic population in regionally dosed, vaccinated animals was most enriched with ERK cascades associated with successful TCR ligation.  

Conclusion

Because tdLNs may be tolerized to cancer, and most cancer treatments involve their resection or irradiation, Ag-directed ICI therapy to non-tdLNs could increase the response rate, potentially reduce adverse events, and increase the number of cancer patients who may benefit from ICI therapy. Supported by CPRIT RP190019 and NIH R21CA267263, R01 CA276513.  WL is a predoctoral fellow supported by the Biomedical Informatics, Genomics and Translational Cancer Research Training Program funded by CPRIT RP210045.