Poster CC8 - Modulation of tumor-associated macrophages via TLR agonists, informed by single-cell RNA sequencing of tumor immune microenvironment, leads to regulatory T cell depletion

Introduction: Tumor-associated macrophages (TAMs) and intratumoral regulatory T cells (Tregs) protect solid tumors from immune clearance and reduce the efficacy of immune checkpoint therapies. Previously, we demonstrated that inducing a robust anti-tumor phenotype in TAMs by activating their toll-like receptors (TLRs) re-invigorates antitumor immunity and improves overall survival in mice (Sarkar et al. Front. Immunol. 2024). However, the relative expression levels of such TLRs in TAMs and the effect of re-programming TAMs on intratumoral Tregs remain incompletely understood. Interestingly, other research has shown that the anti-tumor efficacy of CTLA-4 blockade can be improved by intratumoral Treg depletion. Here, we hypothesized that TAMs can be re-programmed by TLR agonists to deplete Tregs via Fcγ receptor-mediated interactions, sensitizing solid tumors to immune checkpoint blockade.

Materials and

Methods: As toll-like receptor (TLR) agonists can program TAMs to an inflammatory state, we scrutinized TLR expression in TAMs using single-cell RNA sequencing. We identified high expression levels of TLR2 (>10-fold higher than other TLRs) in the immune microenvironment of a intradermal MC38 tumors. Based on the expression profile of TLRs, we tested whether TLR2 is a promising therapeutic target to repolarize TAMs to an immuno-supportive phenotype and indirectly increase intratumoral CD8/Treg ratio. Specifically, we examined the ability of a synthetic TLR2 agonist (CU-T12-9) to repolarize TAMs to an inflammatory phenotype. First, the polarization of murine bone marrow-derived macrophages in response to the TLR2 agonist was assessed in vitro via PCR (relevant genes: Il12, Nos2, and Mrc1). For monotherapy, MC38 tumor-bearing mice were treated every other day by peritumoral injection of CU-T12-9. For combination therapy, treatments consisted of vehicle control, an anti-CTLA4 monoclonal antibody, or combination therapy of anti-CTLA4 and CU-T12-9. At set time points, tumor growth was monitored via caliper measurements.

Results, Conclusions, and Discussions: Drug treatment promoted inflammatory TAM re-polarization (increased levels of Il12 and Nos2; lower levels of Mrc1), but it was ineffective as a monotherapy against established MC38 tumors. In contrast, when administered in combined with anti-CTLA4, CU-T12-9 markedly improved therapeutic response rates (Figure 1A–B). Intratumoral depletion of Tregs was reflected in a 2-fold higher abundance of CD8+ T cells to Tregs (Figure 1C–D). Our data support a model that the adjuvant capability of the TLR2 agonist resulted not only from the inflammatory re-programming of TAMs, but also from (a) the resulting increase in FcγRIV expression level in TAMs (assessed via PCR) and (b) antibody-dependent depletion of anti-CTLA-4-bound Tregs by FcγRIV+ TAMs (Figure 1E). Furthermore, tumor development was attenuated in a model of B16.F10 melanoma metastasis to the lung.

Tissue context and local cues affect macrophage identity, activation, and priming. Hence, single-cell RNA sequencing of patient-specific tumor samples may be useful to detect TLR expression levels, and this information will be crucial for selecting specific TLR agonists as adjuvants for immune checkpoint therapy. Our study highlights the appeal and potential applicability of single-cell RNA sequencing in the selection of tissue-specific anti-cancer therapeutic regimens. Finally, our findings demonstrate the ability of TAM repolarization strategies to support anti-CTLA4 checkpoint therapies, potentially via intratumoral Treg depletion.

Acknowledgements (Optional): B.S. would like to thank the Cotswold Foundation for a postdoctoral fellowship. This work was supported, in part, by grants from the NIH, including T32CA079443 (R.W.), R01CA206890 (R.W.), U01CA206997 (R.W.), R01HL131495 (R.W.), and R35GM147184 (C.B.R.).