Abstract 3807: MicroRNA-29a synergizes with PD-1 therapy to regulate anti-tumor immunity

Abstract CD8 T cells initiate potent anti-tumor immune responses. However, tumor cells employ PD-1 ligand, (PD-L1) to obstruct CD8 T cell responses and drive exhausted T cells (TEX) differentiation. TEX are characterized by sub-optimal functionality and inability to efficiently eliminate tumor cells. Checkpoint inhibitor therapy such as anti-PD-1 can partially restore the effector functions of TEX, but the stable epigenetic programs of TEX lead to the loss of reinvigoration and tumor relapse, providing a challenge for the efficacy of checkpoint blockade therapy. We recently demonstrated that miR-29a acts as a key regulator of TEX differentiation, promotes memory-like differentiation and attenuates exhaustion during chronic infection. Importantly, miR-29a favors the expansion of a progenitor TEX subset expressing the transcription factor TCF-1 that responds to immunotherapy. Therefore, we hypothesized that miR-29a can substantially alter TEX differentiation and synergize with checkpoint blockade to enhance anti-tumor immune responses. A well-established mouse model of chronic infection (LCMV clone-13) was used to induce TEX differentiation, with transcriptional and epigenetic profiles similar to TEX from human tumors. We found that combination of miR-29a overexpression and anti-PD-L1 treatment enhanced CD8 T cell expansion > 20 fold, whereas anti-PD-L1 alone marginally increased the number of CD8 T cells, suggesting that miR-29a synergizes with anti-PD-L1 to enhance CD8 T cell responses. Immune reinvigoration induced by anti-PD-L1 therapy is characterized by a partial reduction of inhibitory receptors and increase in effector responses. Instead, miR-29a overexpression combined with anti-PD-L1 promoted memory-like differentiation and long-term persistence of CD8 T cells, without compromising effector functions. Mechanistically, miR-29a synergized with anti-PD-L1 by inducing expression of a key memory-associated transcription factor, TCF-1, and antagonizing the master epigenetic regulator of exhaustion, TOX. To further understand whether miR-29a promotes only expansion of a progenitor TCF1+ subset or fundamentally alters TEX subset differentiation, equal numbers of miR-29a overexpressing and control TCF1+ CD8 T cells were isolated and adoptively transferred to secondary host mice followed by PD-L1 blockade. TCF1+ CD8 T cells with enforced miR-29a expression responded to PD-L1 blockade more efficiently than control TCF+ CD8 T cells. These results suggest that miR-29a fundamentally alters TEX differentiation by regulating key exhaustion-related transcriptional and epigenetic circuits. Therefore, we suggest that miR29a has the potential to be used as a novel therapeutic inducing robust and durable immune responses upon PD-1 therapy. Citation Format: Xuebing Leng, Svetlana Ristin, Christine Rafie, Lance Buchness, Alejandro Villarino, Erietta Stelekati. MicroRNA-29a synergizes with PD-1 therapy to regulate anti-tumor immunity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3807

Reinvigorated exhausted T cells exhibit distinct features within 24 hours after PD-1 therapy

Abstract CD8 T cells are potent cytolytic immune cells. However, persistent antigens during chronic infections and cancers drive differentiation of exhausted T cells (T EX). T EXdo not generate optimal immune protection. Immunotherapy such as anti-PD-L1 provides a partial T EXreinvigoration. Some T EXregain effector functions, enter cell cycle and start proliferation by PD-1 therapy. However, the underlying molecular and epigenetic mechanisms of T EXreinvigoration are not well understood. Moreover, a key unknown question is why only some T EXgain reinvigoration. Here, we used proliferation as a surrogate for T EXreinvigoration. A transgenic mouse model carrying a cell cycle reporter (Fucci) was used. Fucci LCMV-specific CD8 T cells were transferred to mice that were then infected with chronic LCMV to induce exhaustion. Recipients were treated with anti-PD-L1 at d21 p.i. Fucci staining (cell cycle) and CTV dilution (cell division) allowed us to isolate reinvigorated T EXbefore first cell division and ask what molecular signals induce T EXreinvigoration. Thus, we found that T EXreinvigoration starts as early as 24 hours after a single anti-PD-L1. Proliferating cells upon treatment contained an increased progenitor-like population and a reduced terminally exhausted population compared to control, whereas non-proliferating cells were similar between groups. Single cell transcriptomics data further identified unique transcriptional signatures in reinvigorated T EXupon anti-PD-L1 compared to untreated T EXat 48 hours post treatment. Therefore, we identified the molecular signatures of T EXthat have the potential to become reinvigorated upon immunotherapy and suggest novel molecular targets to enhance the potency of PD-1 therapy

T cell intrinsic STAT1 signaling prevents aberrant Th1 responses during acute toxoplasmosis

Infection-induced T cell responses must be properly tempered and terminated to prevent immuno-pathology. Using transgenic mice, we demonstrate that T cell intrinsic STAT1 signaling is required to curb inflammation during acute infection with Toxoplasma gondii. Specifically, we report that mice lacking STAT1 selectively in T cells expel parasites but ultimately succumb to lethal immuno-pathology characterized by aberrant Th1-type responses with reduced IL-10 and increased IL-13 production. We also find that, unlike STAT1, STAT3 is not required for induction of IL-10 or suppression of IL-13 during acute toxoplasmosis. Each of these findings was confirmed in vitro and ChIP-seq data mining showed that STAT1 and STAT3 co-localize at the Il10 locus, as well as loci encoding other transcription factors that regulate IL-10 production, most notably Maf and Irf4. These data advance basic understanding of how infection-induced T cell responses are managed to prevent immuno-pathology and provide specific insights on the anti-inflammatory properties of STAT1, highlighting its role in shaping the character of Th1-type responses

MicroRNA-29a attenuates CD8 T cell exhaustion and induces memory-like CD8 T cells during chronic infection.

CD8 T cells mediate protection against intracellular pathogens and tumors. However, persistent antigen during chronic infections or cancer leads to T cell exhaustion, suboptimal functionality, and reduced protective capacity. Despite considerable work interrogating the transcriptional regulation of exhausted CD8 T cells (TEX), the posttranscriptional control of TEX remains poorly understood. Here, we interrogated the role of microRNAs (miRs) in CD8 T cells responding to acutely resolved or chronic viral infection and identified miR-29a as a key regulator of TEX. Enforced expression of miR-29a improved CD8 T cell responses during chronic viral infection and antagonized exhaustion. miR-29a inhibited exhaustion-driving transcriptional pathways, including inflammatory and T cell receptor signaling, and regulated ribosomal biogenesis. As a result, miR-29a fostered a memory-like CD8 T cell differentiation state during chronic infection. Thus, we identify miR-29a as a key regulator of TEX and define mechanisms by which miR-29a can divert exhaustion toward a more beneficial memory-like CD8 T cell differentiation state