LAG-3 and PD-1 synergize on CD8+ T cells to drive T cell exhaustion and hinder autocrine IFN-γ-dependent anti-tumor immunity.

Overcoming immune-mediated resistance to PD-1 blockade remains a major clinical challenge. Enhanced efficacy has been demonstrated in melanoma patients with combined nivolumab (anti-PD-1) and relatlimab (anti-LAG-3) treatment, the first in its class to be FDA approved. However, how these two inhibitory receptors synergize to hinder anti-tumor immunity remains unknown. Here, we show that CD8+ T cells deficient in both PD-1 and LAG-3, in contrast to CD8+ T cells lacking either receptor, mediate enhanced tumor clearance and long-term survival in mouse models of melanoma. PD-1- and LAG-3-deficient CD8+ T cells were transcriptionally distinct, with broad TCR clonality and enrichment of effector-like and interferon-responsive genes, resulting in enhanced IFN-γ release indicative of functionality. LAG-3 and PD-1 combined to drive T cell exhaustion, playing a dominant role in modulating TOX expression. Mechanistically, autocrine, cell-intrinsic IFN-γ signaling was required for PD-1- and LAG-3-deficient CD8+ T cells to enhance anti-tumor immunity, providing insight into how combinatorial targeting of LAG-3 and PD-1 enhances efficacy.

Autoreactive CD8+ T cells are restrained by an exhaustion-like program that is maintained by LAG3.

Impaired chronic viral and tumor clearance has been attributed to CD8+ T cell exhaustion, a differentiation state in which T cells have reduced and altered effector function that can be partially reversed upon blockade of inhibitory receptors. The role of the exhaustion program and transcriptional networks that control CD8+ T cell function and fate in autoimmunity is not clear. Here we show that intra-islet CD8+ T cells phenotypically, transcriptionally, epigenetically and metabolically possess features of canonically exhausted T cells, yet maintain important differences. This 'restrained' phenotype can be perturbed and disease accelerated by CD8+ T cell-restricted deletion of the inhibitory receptor lymphocyte activating gene 3 (LAG3). Mechanistically, LAG3-deficient CD8+ T cells have enhanced effector-like functions, trafficking to the islets, and have a diminished exhausted phenotype, highlighting a physiological role for an exhaustion program in limiting autoimmunity and implicating LAG3 as a target for autoimmune therapy.

Interferon-γ Drives Treg Fragility to Promote Anti-tumor Immunity.

Regulatory T cells (Tregs) are a barrier to anti-tumor immunity. Neuropilin-1 (Nrp1) is required to maintain intratumoral Treg stability and function but is dispensable for peripheral immune tolerance. Treg-restricted Nrp1 deletion results in profound tumor resistance due to Treg functional fragility. Thus, identifying the basis for Nrp1 dependency and the key drivers of Treg fragility could help to improve immunotherapy for human cancer. We show that a high percentage of intratumoral NRP1+ Tregs correlates with poor prognosis in melanoma and head and neck squamous cell carcinoma. Using a mouse model of melanoma where Nrp1-deficient (Nrp1-/-) and wild-type (Nrp1+/+) Tregs can be assessed in a competitive environment, we find that a high proportion of intratumoral Nrp1-/- Tregs produce interferon-γ (IFNγ), which drives the fragility of surrounding wild-type Tregs, boosts anti-tumor immunity, and facilitates tumor clearance. We also show that IFNγ-induced Treg fragility is required for response to anti-PD1, suggesting that cancer therapies promoting Treg fragility may be efficacious.

A Cre-driven allele-conditioning line to interrogate CD4+ conventional T cells.

CD4+ T cells share common developmental pathways with CD8+ T cells, and upon maturation, CD4+ T conventional T (Tconv) cells lack phenotypic markers that distinguish these cells from FoxP3+ T regulatory cells. We developed a tamoxifen-inducible ThPOKCreERT2.hCD2 line with Frt sites inserted on either side of the CreERT2-hCD2 cassette, and a Foxp3Ametrine-FlpO strain, expressing Ametrine and FlpO in Foxp3+ cells. Breeding these mice resulted in a CD4conviCreERT2-hCD2 line that allows for the specific manipulation of a gene in CD4+ Tconv cells. As FlpO removes the CreERT2-hCD2 cassette, CD4+ Treg cells are spared from Cre activity, which we refer to as allele conditioning. Comparison with an E8IiCreERT2.GFP mouse that enables inducible targeting of CD8+ T cells, and deletion of two inhibitory receptors, PD-1 and LAG-3, in a melanoma model, support the fidelity of these lines. These engineered mouse strains present a resource for the temporal manipulation of genes in CD4+ T cells and CD4+ Tconv cells.

Treg Cells Promote the SREBP1-Dependent Metabolic Fitness of Tumor-Promoting Macrophages via Repression of CD8+ T Cell-Derived Interferon-γ.

Regulatory T (Treg) cells are crucial for immune homeostasis, but they also contribute to tumor immune evasion by promoting a suppressive tumor microenvironment (TME). Mice with Treg cell-restricted Neuropilin-1 deficiency show tumor resistance while maintaining peripheral immune homeostasis, thereby providing a controlled system to interrogate the impact of intratumoral Treg cells on the TME. Using this and other genetic models, we showed that Treg cells shaped the transcriptional landscape across multiple tumor-infiltrating immune cell types. Treg cells suppressed CD8+ T cell secretion of interferon-γ (IFNγ), which would otherwise block the activation of sterol regulatory element-binding protein 1 (SREBP1)-mediated fatty acid synthesis in immunosuppressive (M2-like) tumor-associated macrophages (TAMs). Thus, Treg cells indirectly but selectively sustained M2-like TAM metabolic fitness, mitochondrial integrity, and survival. SREBP1 inhibition augmented the efficacy of immune checkpoint blockade, suggesting that targeting Treg cells or their modulation of lipid metabolism in M2-like TAMs could improve cancer immunotherapy.

Regulatory T Cell Insufficiency in Autoimmune Diabetes Is Driven by Selective Loss of Neuropilin-1 on Intraislet Regulatory T Cells.

Approaches to reverse or limit regulatory T cell (Treg) insufficiency are of great interest for development of immunotherapeutic treatments for autoimmune patients, including type 1 diabetes. Treg insufficiency is heavily implicated in the progression of autoimmune diabetes in the NOD mouse model and is characterized by defects in Treg numbers, development, and/or function. Utilizing a Treg-centric screen, we show that intraislet Tregs have a uniquely dysfunctional phenotype, hallmarked by an almost complete lack of neuropilin-1 (Nrp1), a cell surface receptor required to maintain Treg stability. Intraislet Nrp1- Tregs exhibit hallmark features of fragility, including reduced suppressive capacity, decreased CD73 and Helios, and increased Rorγt and Tbet. Intraislet Nrp1- Tregs also exhibit decreased Foxp3 expression on a per cell basis, suggesting that Nrp1 may also be required for long-term Treg stability. Mechanistically, Treg-restricted augmentation of Nrp1 expression limited the onset of autoimmune diabetes in NOD mice suggesting that Nrp1 critically impacts intraislet Treg function. Transcriptional analysis showed that Nrp1 restoration led to an increase in markers and pathways of TCR signaling, survival, and suppression, and when Nrp1 protein expression is examined by cellular indexing of transcriptomes and epitopes by sequencing, significant differences were observed between Nrp1+ and Nrp1- Tregs in all tissues, particularly in markers of Treg fragility. This translated into substantive differences between Nrp1+ and Nrp1- Tregs that afforded the former with a competitive advantage in the islets. Taken together, these data suggest that maintenance of Nrp1 expression and signaling on Tregs limits diabetes onset and may serve as a strategy to combat Treg insufficiency in autoimmune disease.

Regulatory T Cell-Derived TRAIL Is Not Required for Peripheral Tolerance.

TRAIL (Tnfsf10/TRAIL/CD253/Apo2L) is an important immune molecule that mediates apoptosis. TRAIL can play key roles in regulating cell death in the tumor and autoimmune microenvironments. However, dissecting TRAIL function remains difficult because of the lack of optimal models. We have now generated a conditional knockout (Tnfsf10 L/L) for cell type-specific analysis of TRAIL function on C57BL/6, BALB/c, and NOD backgrounds. Previous studies have suggested a role for TRAIL in regulatory T cell (Treg)-mediated suppression. We generated mice with a Treg-restricted Tnfsf10 deletion and surprisingly found no impact on tumor growth in C57BL/6 and BALB/c tumor models. Furthermore, we found no difference in the suppressive capacity of Tnfsf10-deficient Tregs and no change in function or proliferation of T cells in tumors. We also assessed the role of TRAIL on Tregs in two autoimmune mouse models: the NOD mouse model of autoimmune diabetes and the myelin oligodendrocyte glycoprotein (MOG) C57BL/6 model of experimental autoimmune encephalomyelitis. We found that deletion of Tnfsf10 on Tregs had no effect on disease progression in either model. We conclude that Tregs do not appear to be dependent on TRAIL exclusively as a mechanism of suppression in both the tumor and autoimmune microenvironments, although it remains possible that TRAIL may contribute in combination with other mechanisms and/or in different disease settings. Our Tnfsf10 conditional knockout mouse should prove to be a useful tool for the dissection of TRAIL function on different cell populations in multiple mouse models of human disease.