Background Despite the transformational impact of immune checkpoint blockade (ICB) in melanoma, only approximately half of patients derive long-term survival benefit. T cells with antiviral signatures have the capacity to secrete inflammatory cytokines/chemokines and deliver potent cytotoxic signals ideal in tumor immunity.¹ A promising therapeutic target is agonism of the double-stranded RNA (dsRNA) antiviral sensor RIG-I. The novel RIG-I agonist Stem Loop RNA (SLR)14 enhanced inflammatory cytokine release and improved the control of murine tumors by TILs and other immune cell types.² However, dsRNA-targeting therapeutic strategies like RIG-I agonism have not yet translated into clinical advances for patients. Further, the effects of SLR14 in human tumors are unknown. We tested the hypothesis that SLR14 transforms T cells to a cytotoxic antiviral state in immunologically ‘cold’ human tumor specimens via type-1 interferon.

Methods We obtained 9 surgical resections from primary melanoma tumors and lymph node metastases and made single-cell suspension replicates of tumor and infiltrating immune cell co-cultures. We stimulated with IFNβ, SLR14, αPD-1, αCD3/CD28+αPD-1 or αCD3/CD28 for 42–48 hours. We Flourescently Activated Cell Sorted (FACS) live cells and then barcoded for multiplexed single cell sequencing using 10x scRNAseq. To visualize the transcriptional response and assign differentiation trajectories following stimulation, we applied PHATE (potential of heat diffusion for affinity-based transition embedding), which facilitates visualization of state transitions and Slingshot, which defines lineage relationships.

Results Following SLR14 stimulation, this approach revealed shifts in tumor co-culture cell-type proportions (figure 1A) and alterations in the transcriptional phenotypes of stem-like progenitor and terminally differentiated T cell populations, which have recently been described in single cell RNAseq studies.^3–6 RIG-I agonism induced new CD4+ and CD8+ populations not observed in positive or negative control conditions (figure 1B,C). In tumor cells, NK cells and T cells, SLR14 stimulation induced expression of canonical IFN-stimulated genes (figure 1D, bottom). In CD8+ T cells, however, we observed specific programs of activation and survival including CD69 (p=0.00003), and IL2RG (p=0.000006) (figure 1D, top). SLR14-induced stem-like CD8+ T cells maintained high levels of IL7R, similar to unstimulated progenitors, but upregulated CD74 (p=0.00004) and IL2RG—suggesting the potential for inflammatory memory formation.^7–8 Similarly, SLR14-induced antiviral CD4+ T cells without any significant increase in Foxp3+ Tregs.

Conclusions RIG-I agonist SLR14 stimulates tumor infiltrating T cells into antiviral states in tumor-immune co-cultures.

References

1. Jiang X, Muthusamy V, Fedorova O, Kong Y, Kim DJ, Bosenberg M, et al. Intratumoral delivery of RIG-I agonist SLR14 induces robust antitumor responses. J Exp Med. 2019;216:2854–2868.

2. Philip M, Schietinger A. CD8+ T cell differentiation and dysfunction in cancer. Nat Rev Immunol. 2022;22:209–223.

3. Miller BC, Sen DR, Al Abosy R, Bi K, Virkud YV, LaFleur MW, et al. Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade. Nat Immunol. 2019;20:326–336.

4. Oliveira G, Stromhaug K, Klaeger S, Kula T, Frederick DT, Le PM, et al. Phenotype, specificity and avidity of antitumour CD8+ T cells in melanoma. Nature. 2021:1–7.

5. McLane LM, Abdel-Hakeem MS, Wherry EJ. CD8 T Cell Exhaustion During Chronic Viral Infection and Cancer. Annu Rev Immunol. 2019. doi:10.1146/annurev-immunol-041015–055318

6. Richer MJ, Pewe LL, Hancox LS, Hartwig SM, Varga SM, Harty JT. Inflammatory IL-15 is required for optimal memory T cell responses. J Clin Invest. 2015;125:3477–3490.

7. Naik S, Larsen SB, Gomez NC, Alaverdyan K, Sendoel A, Yuan S, et al. Inflammatory memory sensitizes skin epithelial stem cells to tissue damage. Nature. 2017;550:475–480.

8. Doherty EH, Piecychna M, Leng L, Bucala R. Adoptive transfer of a novel MIF receptor (CD74+) expressing memory T cell subpopulation is sufficient to transfer inflammatory arthritis. The Journal of Immunology. 2017;198:156.3–156.3.

Ethics Approval Tumors were collected with the approval of the Yale University Institutional Review Board, IRB # 0609001869, approval date: 7/19/2022, and expiration Date: 7/18/2023. Participants gave informed consent before taking part.

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Abstract 1107 Figure 1