Faculty Bibliography

Cancer cells activate the integrated stress response (ISR) to adapt to stress and resist therapy¹. ISR signals converge on activating transcription factor 4 (ATF4), which controls cell-intrinsic transcriptional programs that are involved in metabolic adaptation, survival and growth^2,3. However, whether the ISR-ATF4 axis influences anti-tumour immune responses remains mostly unknown. Here we show that loss of ATF4 decreases tumour progression considerably in immunocompetent mice, but not in immunocompromised ones, by enhancing T cell-dependent anti-cancer immune responses. An unbiased genetic screen of ATF4-regulated genes identifies lipocalin 2 (LCN2) as the principal ATF4-dependent effector that impairs anti-tumour immunity by favouring infiltration with immunosuppressive interstitial macrophages. Furthermore, we find that LCN2 promotes T cell exclusion and immune evasion in preclinical mouse models, and correlates with decreased T cell infiltration in patients with lung and pancreatic adenocarcinomas. Anti-LCN2 antibodies promote robust anti-tumour T cell responses in mouse models of aggressive solid tumours. Our study shows that the ATF4-LCN2 axis has a cell-extrinsic role in suppressing anti-cancer immunity, and could pave the way for an immunotherapy approach that targets LCN2.

Acute myeloid leukemia (AML) is an aggressive myeloid malignancy with a poor prognosis. Venetoclax (Ven), a BCL2 inhibitor, has shown promising results but often leads to relapse due to mitochondrial dysregulation, particularly due to upregulation of the anti-apoptotic protein MCL1. Overexpression of the transcription factor STAT3 has been linked to poor survival in AML patients. Overexpression of STAT3 in a transgenic murine model induces a myeloid malignancy with a short latency period and inflammatory upregulation. The current study identifies STAT3 upregulation as a key mechanism of Ven resistance. A clinically relevant STAT3 degrader effectively reduces both total and phosphorylated STAT3, corrects mitochondrial structural and functional dysregulation, and induces apoptosis in Ven-resistant AML cell lines. KT-333 significantly decreases STAT3 and MCL1 protein levels and improves survival in Ven-resistant (Ven-Res) AML murine models. In summary, STAT3 hyperactivation is leukemogenic, is further potentiated in Ven-resistance and can be clinically targeted with a novel and specific STAT3 degrader. Pictorial representation depicting upregulation of STAT3 and MCL1 in venetoclax resistant myeloid malignancies such as MDS and AML causing mitochondrial structural abnormalities and dysfunction. By using specific STAT3 degrader, STAT3 inhibition, and thereby indirect downregulation of MCL1 can be a promising therapeutic intervention to target drug resistant clones in MDS and AML.

Plasmacytoid dendritic cells (pDCs) mount powerful antiviral type I interferon (IFN-I) responses, yet only a fraction of pDCs produces high levels of IFN-I. Here we report that peripheral pDCs in naive mice comprise three subsets (termed A, B and C) that represent progressive differentiation stages. This heterogeneity was generated by tonic IFN-I signaling elicited in part by the cGAS/STING and TLR9 DNA-sensing pathways. A small 'IFN-I-naive' subset (pDC-A) could give rise to other subsets; it was expanded in STING deficiency or after the IFN-I receptor blockade, but was abolished by exogenous IFN-I. In response to RNA viruses, pDC-A showed increased Bcl2-dependent survival and superior IFN-I responses, but was susceptible to virus infection. Conversely, the majority of pDCs comprised the 'IFN-I-primed' subsets (pDC-B/C) that showed lower IFN-I responses and poor survival, but did not support virus replication. Thus, tonic IFN-I signaling decreases the cytokine-producing capacity and survival of pDCs but increases their virus resistance, facilitating optimal antiviral responses.

UNLABELLED:Evolution and outgrowth of drug-resistant cancer cells are common causes of treatment failure. Patients with leukemic cutaneous T-cell lymphoma have a poor prognosis because of the development of drug resistance and severe bacterial infections. In this study, we show that most patients with leukemic cutaneous T-cell lymphoma harbor multiple genetically distinct subclones that express an identical clonal antigen receptor but display distinct phenotypes and functional properties. These coexisting malignant subclones exhibit differences in tissue homing, metabolism, and cytokine expression and respond differently to extrinsic factors like Staphylococcus aureus and cancer drugs. Indeed, although S. aureus toxins selectively enhance activation and proliferation of certain subclones, these responsive subclones are also the most intrinsically sensitive to cancer drugs when the stimuli are removed. Consequently, although the divergent evolution of malignant subclones drives aggressiveness, adaptability, and drug resistance by removing extrinsic stimuli and mapping malignant subclones, we can expose inherent vulnerabilities that can be exploited in the treatment of these cancers. SIGNIFICANCE/UNASSIGNED:Cancer cells have inherent disparity in hallmark traits, such as aggressiveness and intrinsic drug resistance. We show that segregation of hallmark traits on different coexisting subclones is common and augments adaptability, aggressiveness, and drug resistance of the overall cancer population. Importantly, this segregation exposes vulnerabilities that can be exploited in individualized therapies.

Persistent viral infections are associated with long-term health issues and prolonged transmission. How external perturbations after initial exposure affect the duration of infection is unclear. Here we discovered that murine astrovirus, an enteric RNA virus, persists indefinitely when mice remain unperturbed but is cleared rapidly after cage change. In addition to eliminating the external viral reservoir, cage change also induced interferon-stimulated genes in the intestinal epithelium that are necessary for viral clearance. We further identified that displacing infected animals initially caused a temporary period of immune suppression through the stress hormone corticosterone, which was followed by an immune rebound characterized by activation of CD8 T cells responsible for downstream epithelial antiviral responses. Our findings show how viral persistence can be disrupted by preventing re-exposure and activating immunity upon stress recovery, indicating that external factors can be manipulated to shorten the duration of a viral infection.

Inbred mice used for biomedical research display an underdeveloped immune system compared with adult humans, which is attributed in part to the artificial laboratory environment. Despite representing a central component of adaptive immunity, the impact of the laboratory environment on the B cell compartment has not been investigated in detail. Here, we performed an in-depth examination of B cells following rewilding, the controlled release of inbred laboratory mice into an outdoor enclosure. In rewilded mice, we observed B cells in circulation with increased signs of maturation, alongside heightened germinal center responses within secondary lymphoid organs. Rewilding also expanded B cells in the gut, which was accompanied by elevated systemic levels of immunoglobulin G (IgG) and IgM antibodies reactive to the microbiota. Our findings indicate that exposing laboratory mice to a more natural environment enhances B cell development to better reflect the immune system of free-living mammals.

Hidradenitis suppurativa (HS) is a chronic, debilitating inflammatory skin disease characterized by keratinized epithelial tunnels that grow deeply into the dermis. Here, we examined the immune microenvironment within human HS lesions. Multi-omics profiling and multiplexed imaging identified tertiary lymphoid structures (TLSs) near HS tunnels. These TLSs were enriched with proliferative T cells, including follicular helper (Tfh), regulatory (Treg), and pathogenic T cells (IL17A+ and IFNG+), alongside extensive clonal expansion of plasma cells producing antibodies reactive to keratinocytes. HS fibroblasts express CXCL13 or CCL19 in response to immune cytokines. Using a microfluidic system to mimic TLS on a chip, we found that HS fibroblasts critically orchestrated lymphocyte aggregation via tumor necrosis factor alpha (TNF-α)-CXCL13 and TNF-α-CCL19 feedback loops with B and T cells, respectively; early TNF-α blockade suppressed aggregate initiation. Our findings provide insights into TLS formation in the skin, suggest therapeutic avenues for HS, and reveal mechanisms that may apply to other autoimmune settings, including Crohn's disease.

Cutaneous T-cell lymphoma is characterized by malignant T cells proliferating in a unique tumor microenvironment dominated by keratinocytes (KCs). Skin colonization and infection by Staphylococcus aureus are a common cause of morbidity and are suspected of fueling disease activity. In this study, we show that expression of HLA-DRs, high-affinity receptors for staphylococcal enterotoxins (SEs), by KCs correlates with IFN-γ expression in the tumor microenvironment. Importantly, IFN-γ induces HLA-DR, SE binding, and SE presentation by KCs to malignant T cells from patients with Sézary syndrome and malignant and nonmalignant T-cell lines derived from patients with Sézary syndrome and mycosis fungoides. Likewise, preincubation of KCs with supernatant from patient-derived SE-producing S aureus triggers proliferation in malignant T cells and cytokine release (including IL-2), when cultured with nonmalignant T cells. This is inhibited by pretreatment with engineered bacteriophage S aureus-specific endolysins. Furthermore, alteration in the HLA-DR-binding sites of SE type A and small interfering RNA-mediated knockdown of Jak3 and IL-2Rγ block induction of malignant T-cell proliferation. In conclusion, we show that upon exposure to patient-derived S aureus and SE, KCs stimulate IL-2Rγ/Jak3-dependent proliferation of malignant and nonmalignant T cells in an environment with nonmalignant T cells. These findings suggest that KCs in the tumor microenvironment play a key role in S aureus-mediated disease activity in cutaneous T-cell lymphoma.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer with a ∼50% response rate to immune checkpoint blockade (ICB) therapy. To identify predictive biomarkers, we integrated bulk and single-cell RNA sequencing (RNA-seq) with spatial transcriptomics from a cohort of 186 samples from 116 patients, including bulk RNA-seq from 14 matched pairs pre- and post-ICB. In nonresponders, tumors show evidence of increased tumor proliferation, neuronal stem cell markers, and IL1. Responders have increased type I/II interferons and preexisting tissue resident (Trm) CD8 or Vδ1 γδ T cells that functionally converge with overlapping antigen-specific transcriptional programs and clonal expansion of public T-cell receptors. Spatial transcriptomics demonstrated colocalization of T cells with B and dendritic cells, which supply chemokines and costimulation. Lastly, ICB significantly increased clonal expansion or recruitment of Trm and Vδ1 cells in tumors specifically in responders, underscoring their therapeutic importance. These data identify potential clinically actionable biomarkers and therapeutic targets for MCC. Significance: MCC serves as a model of ICB response. We utilized the largest-to-date, multimodal MCC dataset (n = 116 patients) to uncover unique tumor-intrinsic properties and immune circuits that predict response. We identified CD8 Trm and Vδ1 T cells as clinically actionable mediators of ICB response in major histocompatibility complex-high and -low MCCs, respectively.

The relative and synergistic contributions of genetics and environment to interindividual immune response variation remain unclear, despite implications in evolutionary biology and medicine. Here we quantify interactive effects of genotype and environment on immune traits by investigating C57BL/6, 129S1 and PWK/PhJ inbred mice, rewilded in an outdoor enclosure and infected with the parasite Trichuris muris. Whereas cellular composition was shaped by interactions between genotype and environment, cytokine response heterogeneity including IFNγ concentrations was primarily driven by genotype with consequence on worm burden. In addition, we show that other traits, such as expression of CD44, were explained mostly by genetics on T cells, whereas expression of CD44 on B cells was explained more by environment across all strains. Notably, genetic differences under laboratory conditions were decreased following rewilding. These results indicate that nonheritable influences interact with genetic factors to shape immune variation and parasite burden.