PD-1 cooperates with AIRE-mediated tolerance to prevent lethal autoimmune disease.

Immunological tolerance is established and maintained by a diverse array of safeguards that work together to protect against autoimmunity. Despite the identification of numerous tolerogenic processes, the basis for cooperation among them remains poorly understood. We sought to identify synergy among several well-defined tolerance mediators that alone provide protection only from mild autoimmune symptoms in C57BL/6 mice: BIM, AIRE, CBL-B, and PD-1. Survey of a range of compound mutant mice revealed that the combined loss of the autoimmune regulator, AIRE, with PD-1 unleashed a spontaneous, lethal autoimmune disease. Pdcd1−/−Aire−/− mice succumbed to cachexia before adulthood, with near-complete destruction of the exocrine pancreas. Such fatal autoimmunity was not observed in Pdcd1−/−Bim−/−, Bim−/−Aire−/−, or Cblb−/−Bim−/− mice, suggesting that the cooperation between AIRE-mediated and PD-1­mediated tolerance was particularly potent. Immune profiling revealed largely normal development of FOXP3+ regulatory T (Treg) cells in Pdcd1−/−Aire−/− mice, yet excessive, early activation of effector T cells. Adoptive transfer experiments demonstrated that autoimmune exocrine pancreatitis was driven by conventional CD4+ T cells and could not be prevented by the cotransfer of Treg cells from wild-type mice. The development of autoimmunity in mixed bone marrow chimeras supported these observations, indicating that failure of recessive tolerance was responsible for disease. These findings reveal a potent tolerogenic axis between AIRE and PD-1 that has implications for our understanding of how immune checkpoint blockade might synergize with subclinical defects in central tolerance to elicit autoimmune disease.

Caspase-8 has dual roles in regulatory T cell homeostasis balancing immunity to infection and collateral inflammatory damage.

Targeting the potent immunosuppressive properties of FOXP3+ regulatory T cells (Tregs) has substantial therapeutic potential for treating autoimmune and inflammatory diseases. Yet, the molecular mechanisms controlling Treg homeostasis, particularly during inflammation, remain unclear. We report that caspase-8 is a central regulator of Treg homeostasis in a context-specific manner that is decisive during immune responses. In mouse genetic models, targeting caspase-8 in Tregs led to accumulation of effector Tregs resistant to apoptotic cell death. Conversely, inflammation induced the MLKL-dependent necroptosis of caspase-8-deficient lymphoid and tissue Tregs, which enhanced immunity to a variety of chronic infections to promote clearance of viral or parasitic pathogens. However, improved immunity came at the risk of lethal inflammation in overwhelming infections. Caspase-8 inhibition using a clinical-stage compound revealed that human Tregs have heightened sensitivity to necroptosis compared with conventional T cells. These findings reveal a fundamental mechanism in Tregs that could be targeted to manipulate the balance between immune tolerance versus response for therapeutic benefit.

RNF41 regulates the damage recognition receptor Clec9A and antigen cross-presentation in mouse dendritic cells.

The dendritic cell receptor Clec9A facilitates processing of dead cell-derived antigens for cross-presentation and the induction of effective CD8+ T cell immune responses. Here, we show that this process is regulated by E3 ubiquitin ligase RNF41 and define a new ubiquitin-mediated mechanism for regulation of Clec9A, reflecting the unique properties of Clec9A as a receptor specialized for delivery of antigens for cross-presentation. We reveal RNF41 is a negative regulator of Clec9A and the cross-presentation of dead cell-derived antigens by mouse dendritic cells. Intriguingly, RNF41 regulates the downstream fate of Clec9A by directly binding and ubiquitinating the extracellular domains of Clec9A. At steady-state, RNF41 ubiquitination of Clec9A facilitates interactions with ER-associated proteins and degradation machinery to control Clec9A levels. However, Clec9A interactions are altered following dead cell uptake to favor antigen presentation. These findings provide important insights into antigen cross-presentation and have implications for development of approaches to modulate immune responses.

Dual Targeting of CDK4/6 and BCL2 Pathways Augments Tumor Response in Estrogen Receptor-Positive Breast Cancer.

PURPOSE: Although cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors significantly extend tumor response in patients with metastatic estrogen receptor-positive (ER+) breast cancer, relapse is almost inevitable. This may, in part, reflect the failure of CDK4/6 inhibitors to induce apoptotic cell death. We therefore evaluated combination therapy with ABT-199 (venetoclax), a potent and selective BCL2 inhibitor. EXPERIMENTAL DESIGN: BCL2 family member expression was assessed following treatment with endocrine therapy and the CDK4/6 inhibitor palbociclib. Functional assays were used to determine the impact of adding ABT-199 to fulvestrant and palbociclib in ER+ breast cancer cell lines, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models. A syngeneic ER+ mouse mammary tumor model was used to study the effect of combination therapy on the immune system. RESULTS: Triple therapy was well tolerated and produced a superior and more durable tumor response compared with single or doublet therapy. This was associated with marked apoptosis, including of senescent cells, indicative of senolysis. Unexpectedly, ABT-199 resulted in Rb dephosphorylation and reduced G1-S cyclins, most notably at high doses, thereby intensifying the fulvestrant/palbociclib-induced cell-cycle arrest. Interestingly, a CRISPR/Cas9 screen suggested that ABT-199 could mitigate loss of Rb (and potentially other mechanisms of acquired resistance) to palbociclib. ABT-199 did not abrogate the favorable immunomodulatory effects of palbociclib in a syngeneic ER+ mammary tumor model and extended tumor response when combined with anti-PD1 therapy. CONCLUSIONS: This study illustrates the potential for targeting BCL2 in combination with CDK4/6 inhibitors and supports investigation of combination therapy in ER+ breast cancer.

A Phase Ib Dose-Escalation and Expansion Study of the BCL2 Inhibitor Venetoclax Combined with Tamoxifen in ER and BCL2-Positive Metastatic Breast Cancer.

Venetoclax, a potent and selective BCL2 inhibitor, synergizes with endocrine therapy in preclinical models of ER-positive breast cancer. Using a phase Ib 3 + 3 dose-escalation and expansion study design, 33 patients with ER and BCL2-positive metastatic disease (mean prior regimens, 2; range, 0-8) were treated with daily tamoxifen (20 mg) and venetoclax (200-800 mg). Apart from uncomplicated "on-target" lymphopenia, no dose-limiting toxicities or high-grade adverse events were observed in the escalation phase (15 patients), and 800 mg was selected as the recommended phase II dose (RP2D). In the expansion phase (18 patients), few high-grade treatment-related adverse events were observed. For 24 patients treated at the RP2D, the confirmed radiologic response rate was 54% and the clinical benefit rate was 75%. Treatment responses were preempted by metabolic responses (FDG-PET) at 4 weeks and correlated with serial changes in circulating tumor DNA. Radiologic responses (40%) and clinical benefit (70%) were observed in 10 patients with plasma-detected ESR1 mutations. SIGNIFICANCE: In the first clinical study to evaluate venetoclax in a solid tumor, we demonstrate that combining venetoclax with endocrine therapy has a tolerable safety profile and elicits notable activity in ER and BCL2-positive metastatic breast cancer. These findings support further investigation of combination therapy for patients with BCL2-positive tumors.See related commentary by Drago et al., p. 323.This article is highlighted in the In This Issue feature, p. 305.

Synergy between the KEAP1/NRF2 and PI3K Pathways Drives Non-Small-Cell Lung Cancer with an Altered Immune Microenvironment.

The lung presents a highly oxidative environment, which is tolerated through engagement of tightly controlled stress response pathways. A critical stress response mediator is the transcription factor nuclear factor erythroid-2-related factor 2 (NFE2L2/NRF2), which is negatively regulated by Kelch-like ECH-associated protein 1 (KEAP1). Alterations in the KEAP1/NRF2 pathway have been identified in 23% of lung adenocarcinomas, suggesting that deregulation of the pathway is a major cancer driver. We demonstrate that inactivation of Keap1 and Pten in the mouse lung promotes adenocarcinoma formation. Notably, metabolites identified in the plasma of Keap1f/f/Ptenf/f tumor-bearing mice indicate that tumorigenesis is associated with reprogramming of the pentose phosphate pathway. Furthermore, the immune milieu was dramatically changed by Keap1 and Pten deletion, and tumor regression was achieved utilizing immune checkpoint inhibition. Thus, our study highlights the ability to exploit both metabolic and immune characteristics in the detection and treatment of lung tumors harboring KEAP1/NRF2 pathway alterations.

Combined immune checkpoint blockade as a therapeutic strategy for BRCA1-mutated breast cancer.

Immune checkpoint inhibitors have emerged as a potent new class of anticancer therapy. They have changed the treatment landscape for a range of tumors, particularly those with a high mutational load. To date, however, modest results have been observed in breast cancer, where tumors are rarely hypermutated. Because BRCA1-associated tumors frequently exhibit a triple-negative phenotype with extensive lymphocyte infiltration, we explored their mutational load, immune profile, and response to checkpoint inhibition in a Brca1-deficient tumor model. BRCA1-mutated triple-negative breast cancers (TNBCs) exhibited an increased somatic mutational load and greater numbers of tumor-infiltrating lymphocytes, with increased expression of immunomodulatory genes including PDCD1 (PD-1) and CTLA4, when compared to TNBCs from BRCA1-wild-type patients. Cisplatin treatment combined with dual anti-programmed death-1 and anti-cytotoxic T lymphocyte-associated antigen 4 therapy substantially augmented antitumor immunity in Brca1-deficient mice, resulting in an avid systemic and intratumoral immune response. This response involved enhanced dendritic cell activation, reduced suppressive FOXP3+ regulatory T cells, and concomitant increase in the activation of tumor-infiltrating cytotoxic CD8+ and CD4+ T cells, characterized by the induction of polyfunctional cytokine-producing T cells. Dual (but not single) checkpoint blockade together with cisplatin profoundly attenuated the growth of Brca1-deficient tumors in vivo and improved survival. These findings provide a rationale for clinical studies of combined immune checkpoint blockade in BRCA1-associated TNBC.

Linear ubiquitin chain assembly complex coordinates late thymic T-cell differentiation and regulatory T-cell homeostasis.

The linear ubiquitin chain assembly complex (LUBAC) is essential for innate immunity in mice and humans, yet its role in adaptive immunity is unclear. Here we show that the LUBAC components HOIP, HOIL-1 and SHARPIN have essential roles in late thymocyte differentiation, FOXP3+ regulatory T (Treg)-cell development and Treg cell homeostasis. LUBAC activity is not required to prevent TNF-induced apoptosis or necroptosis but is necessary for the transcriptional programme of the penultimate stage of thymocyte differentiation. Treg cell-specific ablation of HOIP causes severe Treg cell deficiency and lethal immune pathology, revealing an ongoing requirement of LUBAC activity for Treg cell homeostasis. These data reveal stage-specific requirements for LUBAC in coordinating the signals required for T-cell differentiation.

DEC-205 is a cell surface receptor for CpG oligonucleotides.

Synthetic CpG oligonucleotides (ODN) have potent immunostimulatory properties exploited in clinical vaccine trials. How CpG ODN are captured and delivered to the intracellular receptor TLR9, however, has been elusive. Here we show that DEC-205, a multilectin receptor expressed by a variety of cells, is a receptor for CpG ODN. When CpG ODN are used as an adjuvant, mice deficient in DEC-205 have impaired dendritic cell (DC) and B-cell maturation, are unable to make some cytokines such as IL-12, and display suboptimal cytotoxic T-cell responses. We reveal that DEC-205 directly binds class B CpG ODN and enhances their uptake. The CpG-ODN binding function of DEC-205 is conserved between mouse and man, although human DEC-205 preferentially binds a specific class B CpG ODN that has been selected for human clinical trials. Our findings identify an important receptor for class B CpG ODN and reveal a unique function for DEC-205.

The dendritic cell receptor Clec9A binds damaged cells via exposed actin filaments.

The immune system must distinguish viable cells from cells damaged by physical and infective processes. The damaged cell-recognition molecule Clec9A is expressed on the surface of the mouse and human dendritic cell subsets specialized for the uptake and processing of material from dead cells. Clec9A recognizes a conserved component within nucleated and nonnucleated cells, exposed when cell membranes are damaged. We have identified this Clec9A ligand as a filamentous form of actin in association with particular actin-binding domains of cytoskeletal proteins. We have determined the crystal structure of the human CLEC9A C-type lectin domain and propose a functional dimeric structure with conserved tryptophans in the ligand recognition site. Mutation of these residues ablated CLEC9A binding to damaged cells and to the isolated ligand complexes. We propose that Clec9A provides targeted recruitment of the adaptive immune system during infection and can also be utilized to enhance immune responses generated by vaccines.