IFN-γ production by functionally reprogrammed Treg cells promotes anti-tumor efficacy of OX40/CD137 bispecific agonist therapy.

Regulatory T (Treg) cells are highly enriched within many tumors and suppress immune responses to cancer. There is intense interest in reprogramming Treg cells to contribute to anti-tumor immunity. OX40 and CD137 are expressed highly on Treg cells, activated and memory T cells, and NK cells. Here, using a novel tetravalent bispecific antibody targeting mouse OX40 and CD137 (FS120m), we show that OX40/CD137 bispecific agonists induce potent anti-tumor immunity partially dependent upon IFN-γ-production by functionally reprogrammed Treg cells. Treatment of tumor-bearing animals with OX40/CD137 bispecific agonists reprograms Treg cells into both fragile Foxp3+ IFN-γ+ cells with decreased suppressive function, and lineage instable Foxp3- IFN-γ+ cells. Treg cell fragility is partially dependent upon IFN-γ signaling, whereas Treg cell instability is associated with reduced IL-2 signaling upon treatment with OX40/CD137 bispecific agonists. Importantly, conditional deletion of Ifng in Foxp3+ Treg cells and their progeny partially reverses the anti-tumor efficacy of OX40/CD137 bispecific agonist therapy, revealing that reprogramming of Treg cells into IFN-γ-producing cells contributes to the efficacy of OX40/CD137 bispecific agonists. These findings provide insights into mechanisms by which bispecific agonist therapies targeting co-stimulatory receptors highly expressed by Treg cells potentiate anti-tumor immunity in mouse models.

Cross-talk between ILC2 and Gata3high Tregs locally constrains adaptive type 2 immunity.

Regulatory T cells (Tregs) control adaptive immunity and restrain type 2 inflammation in allergic disease. Interleukin-33 promotes the expansion of tissue-resident Tregs and group 2 innate lymphoid cells (ILC2s); however, how Tregs locally coordinate their function within the inflammatory niche is not understood. Here, we show that ILC2s are critical orchestrators of Treg function. Using spatial, cellular, and molecular profiling of the type 2 inflamed niche, we found that ILC2s and Tregs engage in a direct (OX40L-OX40) and chemotaxis-dependent (CCL1-CCR8) cellular dialogue that enforces the local accumulation of Gata3high Tregs, which are transcriptionally and functionally adapted to the type 2 environment. Genetic interruption of ILC2-Treg communication resulted in uncontrolled type 2 lung inflammation after allergen exposure. Mechanistically, we found that Gata3high Tregs can modulate the local bioavailability of the costimulatory molecule OX40L, which subsequently controlled effector memory T helper 2 cell numbers. Hence, ILC2-Treg interactions represent a critical feedback mechanism to control adaptive type 2 immunity.

Implantation and Decidualization in PCOS: Unraveling the Complexities of Pregnancy.

Polycystic Ovary Syndrome (PCOS) is a prevalent endocrine disorder in women of reproductive age, affecting 5-15% globally with a large proportion undiagnosed. This review explores the multifaceted nature of PCOS and its impact on pregnancy, including challenges in fertility due to hormonal imbalances and insulin resistance. Despite restoring ovulation pharmacologically, women with PCOS face lower pregnancy rates and higher risks of implantation failure and miscarriage. Our review focuses on the complexities of hormonal and metabolic imbalances that impair endometrial receptivity and decidualization in PCOS. Disrupted estrogen signaling, reduced integrity of endometrial epithelial tight junctions, and insulin resistance impair the window of endometrial receptivity. Furthermore, progesterone resistance adversely affects decidualization. Our review also examines the roles of various immune cells and inflammatory processes in the endometrium, contributing to the condition's reproductive challenges. Lastly, we discuss the use of rodent models in understanding PCOS, particularly those induced by hormonal interventions, offering insights into the syndrome's impact on pregnancy and potential treatments. This comprehensive review underscores the need for advanced understanding and treatment strategies to address the reproductive complications associated with PCOS, emphasizing its intricate interplay of hormonal, metabolic, and immune factors.

Tumour-retained activated CCR7+ dendritic cells are heterogeneous and regulate local anti-tumour cytolytic activity.

Tumour dendritic cells (DCs) internalise antigen and upregulate CCR7, which directs their migration to tumour-draining lymph nodes (dLN). CCR7 expression is coupled to an activation programme enriched in regulatory molecule expression, including PD-L1. However, the spatio-temporal dynamics of CCR7+ DCs in anti-tumour immune responses remain unclear. Here, we use photoconvertible mice to precisely track DC migration. We report that CCR7+ DCs are the dominant DC population that migrate to the dLN, but a subset remains tumour-resident despite CCR7 expression. These tumour-retained CCR7+ DCs are phenotypically and transcriptionally distinct from their dLN counterparts and heterogeneous. Moreover, they progressively downregulate the expression of antigen presentation and pro-inflammatory transcripts with more prolonged tumour dwell-time. Tumour-residing CCR7+ DCs co-localise with PD-1+CD8+ T cells in human and murine solid tumours, and following anti-PD-L1 treatment, upregulate stimulatory molecules including OX40L, thereby augmenting anti-tumour cytolytic activity. Altogether, these data uncover previously unappreciated heterogeneity in CCR7+ DCs that may underpin a variable capacity to support intratumoural cytotoxic T cells.

Acquisition of suppressive function by conventional T cells limits antitumor immunity upon Treg depletion.

Regulatory T (Treg) cells contribute to immune homeostasis but suppress immune responses to cancer. Strategies to disrupt Treg cell-mediated cancer immunosuppression have been met with limited clinical success, but the underlying mechanisms for treatment failure are poorly understood. By modeling Treg cell-targeted immunotherapy in mice, we find that CD4+ Foxp3- conventional T (Tconv) cells acquire suppressive function upon depletion of Foxp3+ Treg cells, limiting therapeutic efficacy. Foxp3- Tconv cells within tumors adopt a Treg cell-like transcriptional profile upon ablation of Treg cells and acquire the ability to suppress T cell activation and proliferation ex vivo. Suppressive activity is enriched among CD4+ Tconv cells marked by expression of C-C motif receptor 8 (CCR8), which are found in mouse and human tumors. Upon Treg cell depletion, CCR8+ Tconv cells undergo systemic and intratumoral activation and expansion, and mediate IL-10-dependent suppression of antitumor immunity. Consequently, conditional deletion of Il10 within T cells augments antitumor immunity upon Treg cell depletion in mice, and antibody blockade of IL-10 signaling synergizes with Treg cell depletion to overcome treatment resistance. These findings reveal a secondary layer of immunosuppression by Tconv cells released upon therapeutic Treg cell depletion and suggest that broader consideration of suppressive function within the T cell lineage is required for development of effective Treg cell-targeted therapies.

BACH2 restricts NK cell maturation and function, limiting immunity to cancer metastasis.

Natural killer (NK) cells are critical to immune surveillance against infections and cancer. Their role in immune surveillance requires that NK cells are present within tissues in a quiescent state. Mechanisms by which NK cells remain quiescent in tissues are incompletely elucidated. The transcriptional repressor BACH2 plays a critical role within the adaptive immune system, but its function within innate lymphocytes has been unclear. Here, we show that BACH2 acts as an intrinsic negative regulator of NK cell maturation and function. BACH2 is expressed within developing and mature NK cells and promotes the maintenance of immature NK cells by restricting their maturation in the presence of weak stimulatory signals. Loss of BACH2 within NK cells results in accumulation of activated NK cells with unrestrained cytotoxic function within tissues, which mediate augmented immune surveillance to pulmonary cancer metastasis. These findings establish a critical function of BACH2 as a global negative regulator of innate cytotoxic function and tumor immune surveillance by NK cells.

Implications of incidental findings from lung screening for primary care: data from a UK pilot.

Regional lung cancer screening (LCS) is underway in England, involving a "lung health check" (LHC) and low-dose CT scan for those at high risk of cancer. Incidental findings from LHCs or CTs are usually referred to primary care. We describe the proportion of participants referred from the West London LCS pilot to primary care, the indications for referral, the number of general practitioner (GP) attendances and consequent changes to patient management, and provide an estimated cost-burden analysis for primary care. A small proportion (163/1542, 10.6%) of LHC attendees were referred to primary care, primarily for suspected undiagnosed chronic obstructive pulmonary disease (55/163, 33.7%) or for QRISK® (63/163, 38.7%) assessment. Ninety one of 159 (57.2%) participants consenting to follow-up attended GP appointments; costs incurred by primary care were estimated at £5.69/LHC participant. Patient management changes occurred in only 36/159 (22.6%) referred participants. LHCs result in a small increase to primary care workload provided a strict referral protocol is adhered to. Changes to patient management arising from incidental findings referrals are infrequent.

CCR8 marks highly suppressive Treg cells within tumours but is dispensable for their accumulation and suppressive function.

CD4+ regulatory T (Treg) cells, dependent upon the transcription factor Foxp3, contribute to tumour immunosuppression but are also required for immune homeostasis. There is interest in developing therapies that selectively target the immunosuppressive function of Treg cells within tumours without disrupting their systemic anti-inflammatory function. High levels of expression of chemokine (C-C motif) receptor 8 (CCR8) discriminate Treg cells within tumours from those found in systemic lymphoid tissues. It has recently been proposed that disruption of CCR8 function using blocking anti-CCR8 antibodies results in reduced accumulation of Treg cells within tumours and disruption of their immunosuppressive function. Here, using Ccr8-/- mice, we show that CCR8 function is not required for Treg cell accumulation or immunosuppression in the context of syngeneic MC38 colorectal adenocarcinoma and B16 melanoma tumours. We observed high levels of CCR8 expression on tumour-infiltrating Treg cells which were abolished in Ccr8-/- mice. High levels of CCR8 marked cells with high levels of suppressive function. However, whereas systemic ablation of Treg cells resulted in strikingly diminished tumour burden, growth of subcutaneously implanted tumours was unaffected by systemic CCR8 loss. Consistently, we observed minimal impact of systemic CCR8 ablation on the frequency, phenotype and function of tumour-infiltrating Treg cells and conventional T (Tconv) function. These findings suggest that CCR8 is not required for Treg cell accumulation and immunosuppressive function within tumours and that depletion of CCR8+ Treg cells rather than blockade of CCR8 function is a more promising avenue for selective immunotherapy.

A cell-based bioluminescence assay reveals dose-dependent and contextual repression of AP-1-driven gene expression by BACH2.

Whereas effector CD4+ and CD8+ T cells promote immune activation and can drive clearance of infections and cancer, CD4+ regulatory T (Treg) cells suppress their function, contributing to both immune homeostasis and cancer immunosuppression. The transcription factor BACH2 functions as a pervasive regulator of T cell differentiation, promoting development of CD4+ Treg cells and suppressing the effector functions of multiple effector T cell (Teff) lineages. Here, we report the development of a stable cell-based bioluminescence assay of the transcription factor activity of BACH2. Tetracycline-inducible BACH2 expression resulted in suppression of phorbol 12-myristate 13-acetate (PMA)/ionomycin-driven activation of a luciferase reporter containing BACH2/AP-1 target sequences from the mouse Ifng + 18k enhancer. BACH2 expression repressed the luciferase signal in a dose-dependent manner but this activity was abolished at high levels of AP-1 signalling, suggesting contextual regulation of AP-1 driven gene expression by BACH2. Finally, using the reporter assay developed, we find that the histone deacetylase 3 (HDAC3)-selective inhibitor, RGFP966, inhibits BACH2-mediated repression of signal-driven luciferase expression. In addition to enabling mechanistic studies, this cell-based reporter may enable identification of small molecule agonists or antagonists of BACH2 function for drug development.

BACH2 drives quiescence and maintenance of resting Treg cells to promote homeostasis and cancer immunosuppression.

Regulatory T (Treg) cell populations are composed of functionally quiescent resting Treg (rTreg) cells which differentiate into activated Treg (aTreg) cells upon antigen stimulation. How rTreg cells remain quiescent despite chronic exposure to cognate self- and foreign antigens is unclear. The transcription factor BACH2 is critical for early Treg lineage specification, but its function following lineage commitment is unresolved. Here, we show that BACH2 is repurposed following Treg lineage commitment and promotes the quiescence and long-term maintenance of rTreg cells. Bach2 is highly expressed in rTreg cells but is down-regulated in aTreg cells and during inflammation. In rTreg cells, BACH2 binds to enhancers of genes involved in aTreg differentiation and represses their TCR-driven induction by competing with AP-1 factors for DNA binding. This function promotes rTreg cell quiescence and long-term maintenance and is required for immune homeostasis and durable immunosuppression in cancer. Thus, BACH2 supports a "division of labor" between quiescent rTreg cells and their activated progeny in Treg maintenance and function, respectively.

A distal enhancer at risk locus 11q13.5 promotes suppression of colitis by Treg cells.

Genetic variations underlying susceptibility to complex autoimmune and allergic diseases are concentrated within noncoding regulatory elements termed enhancers1. The functions of a large majority of disease-associated enhancers are unknown, in part owing to their distance from the genes they regulate, a lack of understanding of the cell types in which they operate, and our inability to recapitulate the biology of immune diseases in vitro. Here, using shared synteny to guide loss-of-function analysis of homologues of human enhancers in mice, we show that the prominent autoimmune and allergic disease risk locus at chromosome 11q13.52-7 contains a distal enhancer that is functional in CD4+ regulatory T (Treg) cells and required for Treg-mediated suppression of colitis. The enhancer recruits the transcription factors STAT5 and NF-κB to mediate signal-driven expression of Lrrc32, which encodes the protein glycoprotein A repetitions predominant (GARP). Whereas disruption of the Lrrc32 gene results in early lethality, mice lacking the enhancer are viable but lack GARP expression in Foxp3+ Treg cells, which are unable to control colitis in a cell-transfer model of the disease. In human Treg cells, the enhancer forms conformational interactions with the promoter of LRRC32 and enhancer risk variants are associated with reduced histone acetylation and GARP expression. Finally, functional fine-mapping of 11q13.5 using CRISPR-activation (CRISPRa) identifies a CRISPRa-responsive element in the vicinity of risk variant rs11236797 capable of driving GARP expression. These findings provide a mechanistic basis for association of the 11q13.5 risk locus with immune-mediated diseases and identify GARP as a potential target in their therapy.

Curability and transferability of atopy with allogeneic hematopoietic cell transplantation.

Atopy is excessive production of IgE in response to allergens. We evaluated in patients undergoing allogeneic hematopoietic cell transplantation (HCT) the following hypotheses: (1) Atopy is "curable" in atopic patients receiving HCT from a nonatopic donor (D-R+), and (2) Atopy is transferable from atopic donors to nonatopic recipients (D+R-). Atopic patients with atopic donors (D+R+) and non-atopic patients with non-atopic donors (D-R-) served as controls. We measured levels of multiallergen-specific IgE (A-IgE, atopy defined as ≥0.35 kUA/L) in sera from 54 patients and their donors pre HCT and from the patients at ≥2 years post HCT. Only 7/12 (58%) D- R+ patients became nonatopic after HCT. Only 1/11 (9%) D+R- patients became atopic. Eleven of 13 (85%) D-R- patients remained nonatopic. Unexpectedly, 11/18 (61%) D+R+ patients became nonatopic. In conclusion, contrary to our hypothesis and previous reports, the "cure" of atopy may occur in only some D-R+ patients and the transfer of atopy may occur rarely. The "cure" may not be necessarily due to the exchange of atopic for nonatopic immune system, as the "cure" may also occur in D+R+ patients.

Bystander T Cells: A Balancing Act of Friends and Foes.

T cell responses are essential for appropriate protection against pathogens. T cell immunity is achieved through the ability to discriminate between foreign and self-molecules, and this relies heavily on stringent T cell receptor (TCR) specificity. Recently, bystander activated T lymphocytes, that are specific for unrelated epitopes during an antigen-specific response, have been implicated in diverse diseases. Numerous infection models have challenged the classic dogma of T cell activation as being solely dependent on TCR and major histocompatibility complex (MHC) interactions, indicating an unappreciated role for pathogen-associated receptors on T cells. We discuss here the specific roles of bystander activated T cells in pathogenesis, shedding light on the ability of these cells to modulate disease severity independently from TCR recognition.

Influence of Chemotherapy on Allergen-Specific IgE.

BACKGROUND: Atopy is defined as excess allergen-specific IgE (A-IgE). IgE is produced by plasma cells that differentiate from allergen-specific B cells. B cells are known to be killed by chemotherapy; however, it is not known whether A-IgE-secreting plasma cells are killed or inhibited by chemotherapy. If yes, serum A-IgE levels would be expected to decrease after chemotherapy. OBJECTIVES: We aimed to determine whether A-IgE levels in atopic individuals (serum A-IgE ≥0.35 kUA/L) decrease into the nonatopic range (< 0.35 kUA/L) after chemotherapy. METHODS: In 105 patients undergoing chemotherapy for acute leukemia, we measured serum A-IgE before and after chemotherapy. In a subset of these patients, we also measured B cell counts before and after chemotherapy. RESULTS: Of the 105 patients, 36 were atopic. In these patients, median A-IgE level before chemotherapy was 1.6 kUA/L whereas the median level after chemotherapy was 0.6 kUA/L (p < 0.001). In 12/36 (33%) patients, A-IgE levels decreased into the nonatopic range. In nonatopic patients (n = 69), the median A-IgE level also dropped: from 0.04 kUA/L before to 0.03 kUA/L after chemotherapy (p = 0.001). Among the total patients (n = 105), the median pre:post-chemotherapy A-IgE ratio was 1.8 (2.6 in atopic and 1.5 in nonatopic patients). In contrast, the median ratio of pre:post-chemotherapy B cell counts was 87.6. CONCLUSIONS: A-IgE levels decrease after chemotherapy but markedly less than B cell counts. Thus, at least some A-IgE plasma cells appear to survive chemotherapy.

IL-10 Deficiency Reveals a Role for TLR2-Dependent Bystander Activation of T Cells in Lyme Arthritis.

T cells predominate the immune responses in the synovial fluid of patients with persistent Lyme arthritis; however, their role in Lyme disease remains poorly defined. Using a murine model of persistent Lyme arthritis, we observed that bystander activation of CD4+ and CD8+ T cells leads to arthritis-promoting IFN-γ, similar to the inflammatory environment seen in the synovial tissue of patients with posttreatment Lyme disease. TCR transgenic mice containing monoclonal specificity toward non-Borrelia epitopes confirmed that bystander T cell activation was responsible for disease development. The microbial pattern recognition receptor TLR2 was upregulated on T cells following infection, implicating it as marker of bystander T cell activation. In fact, T cell-intrinsic expression of TLR2 contributed to IFN-γ production and arthritis, providing a mechanism for microbial-induced bystander T cell activation during infection. The IL-10-deficient mouse reveals a novel TLR2-intrinsic role for T cells in Lyme arthritis, with potentially broad application to immune pathogenesis.

Snai2 and Snai3 transcriptionally regulate cellular fitness and functionality of T cell lineages through distinct gene programs.

T lymphocytes are essential contributors to the adaptive immune system and consist of multiple lineages that serve various effector and regulatory roles. As such, precise control of gene expression is essential to the proper development and function of these cells. Previously, we identified Snai2 and Snai3 as being essential regulators of immune tolerance partly due to the impaired function of CD4(+) regulatory T cells in Snai2/3 conditional double knockout mice. Here we extend those previous findings using a bone marrow transplantation model to provide an environmentally unbiased view of the molecular changes imparted onto various T lymphocyte populations once Snai2 and Snai3 are deleted. The data presented here demonstrate that Snai2 and Snai3 transcriptionally regulate the cellular fitness and functionality of not only CD4(+) regulatory T cells but effector CD8(α+) and CD4(+) conventional T cells as well. This is achieved through the modulation of gene sets unique to each cell type and includes transcriptional targets relevant to the survival and function of each T cell lineage. As such, Snai2 and Snai3 are essential regulators of T cell immunobiology.

Host actin polymerization tunes the cell division cycle of an intracellular pathogen.

Growth and division are two of the most fundamental capabilities of a bacterial cell. While they are well described for model organisms growing in broth culture, very little is known about the cell division cycle of bacteria replicating in more complex environments. Using a D-alanine reporter strategy, we found that intracellular Listeria monocytogenes (Lm) spend a smaller proportion of their cell cycle dividing compared to Lm growing in broth culture. This alteration to the cell division cycle is independent of bacterial doubling time. Instead, polymerization of host-derived actin at the bacterial cell surface extends the non-dividing elongation period and compresses the division period. By decreasing the relative proportion of dividing Lm, actin polymerization biases the population toward cells with the highest propensity to form actin tails. Thus, there is a positive-feedback loop between the Lm cell division cycle and a physical interaction with the host cytoskeleton.

Pseudomonas aeruginosa-induced bleb-niche formation in epithelial cells is independent of actinomyosin contraction and enhanced by loss of cystic fibrosis transmembrane-conductance regulator osmoregulatory function.

UNLABELLED: The opportunistic pathogen Pseudomonas aeruginosa can infect almost any site in the body but most often targets epithelial cell-lined tissues such as the airways, skin, and the cornea of the eye. A common predisposing factor is cystic fibrosis (CF), caused by defects in the cystic fibrosis transmembrane-conductance regulator (CFTR). Previously, we showed that when P. aeruginosa enters epithelial cells it replicates intracellularly and occupies plasma membrane blebs. This phenotype is dependent on the type 3 secretion system (T3SS) effector ExoS, shown by others to induce host cell apoptosis. Here, we examined mechanisms for P. aeruginosa-induced bleb formation, focusing on its relationship to apoptosis and the CFTR. The data showed that P. aeruginosa-induced blebbing in epithelial cells is independent of actin contraction and is inhibited by hyperosmotic media (400 to 600 mOsM), distinguishing bacterially induced blebs from apoptotic blebs. Cells with defective CFTR displayed enhanced bleb formation upon infection, as demonstrated using bronchial epithelial cells from a patient with cystic fibrosis and a CFTR inhibitor, CFTR(Inh)-172. The defect was found to be correctable either by incubation in hyperosmotic media or by complementation with CFTR (pGFP-CFTR), suggesting that the osmoregulatory function of CFTR counters P. aeruginosa-induced bleb-niche formation. Accordingly, and despite their reduced capacity for bacterial internalization, CFTR-deficient cells showed greater bacterial occupation of blebs and enhanced intracellular replication. Together, these data suggest that P. aeruginosa bleb niches are distinct from apoptotic blebs, are driven by osmotic forces countered by CFTR, and could provide a novel mechanism for bacterial persistence in the host. IMPORTANCE: Pseudomonas aeruginosa is an opportunistic pathogen problematic in hospitalized patients and those with cystic fibrosis (CF). Previously, we showed that P. aeruginosa can enter epithelial cells and replicate within them and traffics to the membrane blebs that it induces. This "bleb-niche" formation requires ExoS, previously shown to cause apoptosis. Here, we show that the driving force for bleb-niche formation is osmotic pressure, differentiating P. aeruginosa-induced blebs from apoptotic blebs. Either CFTR inhibition or CFTR mutation (as seen in people with CF) causes P. aeruginosa to make more bleb niches and provides an osmotic driving force for blebbing. CFTR inhibition also enhances bacterial occupation of blebs and intracellular replication. Since CFTR is targeted for removal from the plasma membrane when P. aeruginosa invades a healthy cell, these findings could relate to pathogenesis in both CF and healthy patient populations.

(D)-Amino acid chemical reporters reveal peptidoglycan dynamics of an intracellular pathogen.

Peptidoglycan (PG) is an essential component of the bacterial cell wall. Although experiments with organisms in vitro have yielded a wealth of information on PG synthesis and maturation, it is unclear how these studies translate to bacteria replicating within host cells. We report a chemical approach for probing PG in vivo via metabolic labeling and bioorthogonal chemistry. A wide variety of bacterial species incorporated azide and alkyne-functionalized d-alanine into their cell walls, which we visualized by covalent reaction with click chemistry probes. The d-alanine analogues were specifically incorporated into nascent PG of the intracellular pathogen Listeria monocytogenes both in vitro and during macrophage infection. Metabolic incorporation of d-alanine derivatives and click chemistry detection constitute a facile, modular platform that facilitates unprecedented spatial and temporal resolution of PG dynamics in vivo.