An anti-tumor coup: TIM3 ablation activates the immune arsenal

In a recent study published in Nature, Dixon et al. showed that TIM3 adjusts “tumor immune temperature” via the involvement of dendritic cells (DCs) with an altered inflammasome activity. Ablation of TIM3 in DCs primarily escalated inflammasome activation and bolstered stem-like CD8 T cells leading to antitumor immunity. In 2002, the discovery of “T cell immunoglobulin and mucin domain-containing protein 3” (TIM3) as an immunoregulatory transmembrane receptor on T-helper cells had led to multiple translational advancements in the field of chronic viral infections, cancer, and other diseases. The translational bravado of TIM3 in the diseases has been evident after being used in several clinical trials. The remarkable common feature of the studies involving TIM3 targeting was overcoming T-cell dysfunction and exhaustion in anti-tumor and anti-viral immunity. Like in T cells, TIM3 was also present in other cell types such as DCs, myeloid-derived suppressor cells (MDSCs), macrophages, natural killer (NK) cells, and mast cells. Far more than being expressed in the different immune system players, genetic alteration of TIM3 has also been linked to allergic and autoimmune diseases. Sakuishi et al. showed that TIM3PD-1 tumor-infiltrating CD8 T lymphocytes (TILs) constitute a significant portion of T cells residing in tumors in preclinical mouse models. Targeting this overwhelming coupling of TIM3PD-1 depicted drastic tumor regression and reversed these inoperative and severely exhausted CD8 TILs. Following this study, the strategy of TIM3 ablation has suddenly appeared in clinical trials with the anti-PD-1 combination in solid tumors. Interestingly, Dixon et al. recently showed that TIM3 hampers anti-tumor immunity of DCs via altering inflammasome and oxidative stress regulation. Generation of conditional knock-out mice targeting TIM3 in T cells showed that TIM3 influenced tumor growth of immunogenic MC38 colon carcinoma cells (MC38-OVA) only in modest levels with the involvement of both CD4 and CD8 T cell subtypes. These mild changes steered researchers to reveal the function of TIM3 in myeloid cells, especially DCs, dictating anti-tumor immunity. Single-cell RNA sequencing (sc-RNAseq) analysis of TILs in wild-type mice bearing MC38-OVA has illustrated predominant expression of TIM3 on DCs, including DC1s and migratory DCs (migDCs). Tumors have shrunk significantly upon conditional deletion of TIM3 in DCs using CD11c (i.e., Havcr2) (Fig. 1). The antitumor function of TIM3 deletion in DCs was also observed in non-small-cell lung carcinoma. Moreover, the tumor inhibitory effect of TIM3 deletion was superior in the DCs compared to T cells. Although Havcr2 tumors had elevated CD8 T cell infiltration, no noteworthy changes in the levels of cytokines, chemokines, and co-inhibitory and -stimulatory molecules on the DCs were observed. Identifying different cellular clusters in Havcr2 and Havcr2 via sc-RNAseq delineated the increased proportion of gene signatures belonging to the memory precursor CD8 T cells, presenting stem-like features such as increased proliferative activity to be further exploited via immune checkpoint blockade. In Havcr2 mice, they also identified PD-1CD8 TILs expressing more IL-7R, CD5, and CXCR5, pointing to the stem-like features of CD8 TILs. Ablation of TIM3 in DCs promoted the preserved existence of stem-like CD8 T cells, which are therapeutically exploitable (Fig. 1). On the other hand, following TIM3 deletion, scRNAseq revealed another subcluster of myeloid cells named migDCs. migDCs showed weakened expressions of the immunoregulatory mediators, including IL-4R, CD200, CD83, and OX-40. Reversing the tumor growth dynamics in Havcr2 mice via antiIL4 treatment has proven that TIM3 ablation enables an antigenspecific anti-tumor immunity (Fig. 1). What caused these effects in CD8 T cells and migDCs following TIM3 loss? With the help of the Waddington OT package, the cellular interactions between the CD8 T cells and the migDCs were exhibited. Provocatively, Il18Il18r1 and Il18-Il18rap were the ligand-receptor gene pairs showing a significantly increased interaction score. Moreover, Havcr2 mice possessed enriched scores for Il18 and Il1 gene sets in CD8 T cells, allowing researchers to hypothesize an existing link between the loss of TIM3 and inflammasome activation. In parallel, migDCs in Havcr2 mice had significant enrichment for the inflammasome activation. Dixon et al. identified an oxidative stress-associated gene signature in migDCs augmenting inflammasome activation in Havcr2 mice (Fig. 1). Especially, immunogenic colon carcinoma cells bearing Havcr2 mice harbored the increased oxidative stress in DCs. Inhibition of reactive oxygen species via N-acetylcysteine treatment rescued the phenotype of anti-tumor immunity in Havcr2 mice. Last, to understand how inflammasome activation precisely controls anti-tumor immune response in tumor-bearing Havcr2 mice, the Kuchroo team has utilized three different approaches: (1) caspase-1 inhibition, (2) MCC950 treatment to destroy ASC complex, and (3) blockage of IL1β/IL-18 axis with the antibodies which was the most vigorous way to lower the tumor temperature. In the context of cancer therapeutics, targeting TIM3 holds promise as it has already undergone several clinical trials. And yet, from the clinician ́s perspective, critical questions remain. In the era of personalized oncology, markers to stratify patients for such combined therapies are required. Do we have to add more

In a recent study published in Nature, Dixon et al. 1 showed that TIM3 adjusts "tumor immune temperature" via the involvement of dendritic cells (DCs) with an altered inflammasome activity. Ablation of TIM3 in DCs primarily escalated inflammasome activation and bolstered stem-like CD8 + T cells leading to antitumor immunity. 1 In 2002, the discovery of "T cell immunoglobulin and mucin domain-containing protein 3" (TIM3) as an immunoregulatory transmembrane receptor on T-helper cells had led to multiple translational advancements in the field of chronic viral infections, cancer, and other diseases. 2,3 The translational bravado of TIM3 in the diseases has been evident after being used in several clinical trials. The remarkable common feature of the studies involving TIM3 targeting was overcoming T-cell dysfunction and exhaustion in anti-tumor and anti-viral immunity. 3 Like in T cells, TIM3 was also present in other cell types such as DCs, myeloid-derived suppressor cells (MDSCs), macrophages, natural killer (NK) cells, and mast cells. 4 Far more than being expressed in the different immune system players, genetic alteration of TIM3 has also been linked to allergic and autoimmune diseases. 3 Sakuishi et al. showed that TIM3 + PD-1 + tumor-infiltrating CD8 + T lymphocytes (TILs) constitute a significant portion of T cells residing in tumors in preclinical mouse models. 5 Targeting this overwhelming coupling of TIM3 + PD-1 + depicted drastic tumor regression and reversed these inoperative and severely exhausted CD8 + TILs. 5 Following this study, the strategy of TIM3 ablation has suddenly appeared in clinical trials with the anti-PD-1 combination in solid tumors.
Interestingly, Dixon et al. recently showed that TIM3 hampers anti-tumor immunity of DCs via altering inflammasome and oxidative stress regulation. 1 Generation of conditional knock-out mice targeting TIM3 in T cells showed that TIM3 influenced tumor growth of immunogenic MC38 colon carcinoma cells (MC38-OVA dim ) only in modest levels with the involvement of both CD4 + and CD8 + T cell subtypes. These mild changes steered researchers to reveal the function of TIM3 in myeloid cells, especially DCs, dictating anti-tumor immunity. Single-cell RNA sequencing (sc-RNAseq) analysis of TILs in wild-type mice bearing MC38-OVA dim has illustrated predominant expression of TIM3 on DCs, including DC1s and migratory DCs (migDCs). Tumors have shrunk significantly upon conditional deletion of TIM3 in DCs using CD11c cre (i.e., Havcr2 cko ) (Fig. 1). The anti-tumor function of TIM3 deletion in DCs was also observed in non-small-cell lung carcinoma. Moreover, the tumor inhibitory effect of TIM3 deletion was superior in the DCs compared to T cells. Although Havcr2 cko tumors had elevated CD8 + T cell infiltration, no noteworthy changes in the levels of cytokines, chemokines, and co-inhibitory and -stimulatory molecules on the DCs were observed.
Identifying different cellular clusters in Havcr2 fl/fl and Havcr2 cko via sc-RNAseq delineated the increased proportion of gene signatures belonging to the memory precursor CD8 + T cells, presenting stem-like features such as increased proliferative activity to be further exploited via immune checkpoint blockade. In Havcr2 cko mice, they also identified PD-1 + CD8 + TILs expressing more IL-7R, CD5, and CXCR5, pointing to the stem-like features of CD8 + TILs. Ablation of TIM3 in DCs promoted the preserved existence of stem-like CD8 + T cells, which are therapeutically exploitable (Fig. 1). On the other hand, following TIM3 deletion, sc-RNAseq revealed another subcluster of myeloid cells named migDCs. migDCs showed weakened expressions of the immunoregulatory mediators, including IL-4R, CD200, CD83, and OX-40. Reversing the tumor growth dynamics in Havcr2 fl/fl mice via anti-IL4 treatment has proven that TIM3 ablation enables an antigenspecific anti-tumor immunity (Fig. 1). What caused these effects in CD8 + T cells and migDCs following TIM3 loss? With the help of the Waddington OT package, the cellular interactions between the CD8 + T cells and the migDCs were exhibited. Provocatively, Il18-Il18r1 and Il18-Il18rap were the ligand-receptor gene pairs showing a significantly increased interaction score. Moreover, Havcr2 cko mice possessed enriched scores for Il18 and Il1 gene sets in CD8 + T cells, allowing researchers to hypothesize an existing link between the loss of TIM3 and inflammasome activation. In parallel, migDCs in Havcr2 cko mice had significant enrichment for the inflammasome activation. Dixon et al. identified an oxidative stress-associated gene signature in migDCs augmenting inflammasome activation in Havcr2 cko mice (Fig. 1). Especially, immunogenic colon carcinoma cells bearing Havcr2 cko mice harbored the increased oxidative stress in DCs. Inhibition of reactive oxygen species via N-acetylcysteine treatment rescued the phenotype of anti-tumor immunity in Havcr2 cko mice. Last, to understand how inflammasome activation precisely controls anti-tumor immune response in tumor-bearing Havcr2 cko mice, the Kuchroo team has utilized three different approaches: (1) caspase-1 inhibition, (2) MCC950 treatment to destroy ASC complex, and (3) blockage of IL-1β/IL-18 axis with the antibodies which was the most vigorous way to lower the tumor temperature.
In the context of cancer therapeutics, targeting TIM3 holds promise as it has already undergone several clinical trials. And yet, from the clinician´s perspective, critical questions remain. In the era of personalized oncology, markers to stratify patients for such combined therapies are required. Do we have to add more markers to the already existing ones (MSI-H, CPS scores)? Is the treatment timeline starting with the conventional chemotherapy always required to deviate from the immunogenic profile of the tumor and the preparedness of it for the TIM3 blockade? If so, are we aware of differences in chemotherapeutic agents interfering with inflammasome activation or inhibition? Should we simultaneously or sequentially combine TIM3 blockade with checkpoint inhibitors in patients eligible for immune therapy? Is TIM3 blockade a way to overcome resistance towards checkpoint inhibitors? Can TIM-3 modulation ex-and in vivo help dendritic cell vaccine engineering along with an increased therapy response? And yet, TIM3 with its mode of action involving the inflammasome, is a refreshing new player in the emerging field of immune therapy and will stimulate new approaches in immune therapy. The future will show how successful and sustainable such clinical studies will be.
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