Resident Lymphocytes Research Articles

The myocardium of human dilated nonischemic cardiomyopathy harbors unique tissue-resident lymphocyte subsets

Abstract Purpose The etiology of dilated nonischemic cardiomyopathy (NICM) is often unclear, but thought to be multifactorial, involving an interplay between genetic factors and direct myocardial damage caused by infection, autoimmunity, or toxins. Tissue-resident lymphocytes are non-circulating immune cells which are retained within various tissues and play integral roles in maintaining homeostasis, responding to infection, regulating inflammation, and mediating tissue repair. Despite their likely involvement in various cardiac pathologies, the presence of tissue-resident lymphocytes in the human heart has not yet been well-characterized, neither in health nor in disease. Methods Human left ventricular tissue and paired peripheral blood samples were obtained from six adult organ donors with normal heart function who died of noncardiac causes and from the explants of six adult patients with end-stage nonischemic cardiomyopathy. Heart tissue was processed using mechanical and enzymatic digestion. Mononuclear cells were isolated from both heart and paired blood by density centrifugation. Isolated mononuclear cells were surface stained with an antibody cocktail and interrogated using multidimensional flow cytometry. Results The adult human heart contains a diverse immune cell population (Fig 1A). Both healthy and NICM cardiac lymphocytes express distinct phenotypic profiles which resemble those of resident lymphocytes found across various tissues, distinct from those isolated from paired blood. Frequencies of effector memory T (Tem) cells (CD45RA- CCR7-) were higher in heart than in blood among both CD4+ and CD8+ T-cells. In heart compared to blood, significantly more Tem expressed CD69, a canonical marker of tissue-resident memory T (Trm) cells (Fig 1B). Cardiac Trm, especially CD8+ Trm, expressed classical Trm signature markers including integrins CD103 (epithelial binding) and CD49a (collagen binding). NICM Trm expressed more CD49a than healthy heart Trm (Fig 1C). NK-cells also demonstrated higher CD69 expression (trNK) in heart than in blood, with associated expression of CD103, CD49a, and CXCR6 (Fig 1D). Compared to healthy hearts, NICM trNK were predominantly CD56bright/CD16- (Fig 1E and 1F) and expressed less CD57 (Fig 1D), overall indicating a less mature state with lower cytotoxic potential. Confocal imaging of cardiac tissue revealed CD69-expressing tissue-resident lymphocytes within the cardiac interstitium. Conclusions The adult human heart contains diverse populations of lymphocytes which express canonical markers of tissue residency, similar to lymphocytes resident in other tissues. Compared to healthy hearts, NICM hearts have more CD49a+ Trm, perhaps related to greater fibrosis, and also harbor a distinct subset of immature tissue-resident NK cells which may shed light on pathophysiology. Future work is focused on better exploring the functional phenotypes of these resident cells and better localizing them within the myocardium.FA - Figure 1

Immunohistochemical Expression of Immune Regulatory Proteins in Interface Dermatoses.

Cutaneous immune-related adverse events (irAEs) of immunotherapies, such as anti-programmed cell death protein-1 (PD-1), suggest that immune checkpoint factors may contribute to the pathobiology of lichenoid interface dermatitis in immunotherapy-naïve patients. Our study aimed to describe innate and adaptive immune markers via immunohistochemical (IHC) staining of lichenoid interface dermatoses. We studied the staining patterns of PD-L1, STING, IL-36 gamma, CD8, PD-1, and LAG-3 in five interface dermatoses: oral lichen planus (LP) (n = 10), cutaneous LP (n = 10), chronic cutaneous lupus erythematosus (CLE) (n = 11), erythema multiforme (EM) (n = 11), and toxic epidermal necrolysis (TEN) (n = 13), by immunohistochemistry (IHC) analysis. Expression was evaluated semi-quantitively according to the percentage of keratinocytes and dermal lymphocytes stained compared to keratinocytes and resident pericapillary lymphocytes in normal human skin. All interface dermatoses evaluated showed increased expression of PD-L1 on keratinocytes and LAG-3 in lymphocytes. STING was increased on the keratinocytes of most specimens. Expression of IL-36 gamma, in basal layer keratinocytes was more extensive in oral LP and cutaneous LP and varied in CLE, EM, and TEN. Lymphocytic infiltration expressing PD-1 was elevated in oral LP, cutaneous LP, and CLE. Current thinking is that interface dermatitis is the result of a cell-mediated immune reaction involving cytotoxic CD8+ T-cell-mediated apoptosis of keratinocytes. The findings of this study suggest that in addition to cell-mediated immunity, innate immune factors may contribute to pathobiology.

Innate lymphoid cells and infectious diseases.

Innate lymphoid cells (ILCs) are the main resident lymphocytes that mostly reside in tissues owing to the lack of adaptive antigen receptors. These cells are involved in early anti-infective immunity, antitumour immunity, regulation of tissue inflammation, and maintenance of homeostasis in the internal environment of tissues and have been referred to as the "first armies stationed in the human body". ILCs are widely distributed in the lungs, colon, lymph nodes, oral mucosa and even embryonic tissues. Due to the advantage of their distribution location, they are often among the first cells to come into contact with pathogens.Relevant studies have demonstrated that ILCs play an early role in the defence against a variety of pathogenic microorganisms, including bacteria, viruses, fungi and helminths, before they intervene in the adaptive immune system. ILCs can initiate a rapid, nonspecific response against pathogens prior to the initiation of an adaptive immune response and can generate a protective immune response against specific pathogens, secreting different effectors to play a role.There is growing evidence that ILCs play an important role in host control of infectious diseases. In this paper, we summarize and discuss the current known infectious diseases in which ILCs are involved and ILC contribution to the defence against infectious diseases. Further insights into the mechanisms of ILCs action in different infectious diseases will be useful in facilitating the development of therapeutic strategies for early control of infections.

Mammalian Inner Ear-Resident Immune Cells-A Scoping Review.

Several studies have demonstrated the presence of resident immune cells in the healthy inner ear. This scoping review aimed to systematize this knowledge by collecting the data on resident immune cells in the inner ear of different species under steady-state conditions. The databases PubMed, MEDLINE (Ovid), CINAHL (EBSCO), and LIVIVO were used to identify articles. Systematic reviews, experimental studies, and clinical data in English and German were included without time limitations. The search yielded 49 eligible articles published between 1979 and 2022. Resident immune cells, including macrophages, lymphocytes, leukocytes, and mast cells, have been observed in various mammalian inner ear structures under steady-state conditions. However, the physiological function of these cells in the healthy cochlea remains unclear, providing an opportunity for basic research in inner ear biology. This review highlights the need for further investigation into the role of these cells, which is crucial for advancing the development of therapeutic methods for treating inner ear disorders, potentially transforming the field of otolaryngology and immunology.

B cells in the pneumococcus-infected lung are heterogeneous and require CD4+ T cell help including CD40L to become resident memory B cells.

Recovery from respiratory pneumococcal infections generates lung-localized protection against heterotypic bacteria, mediated by resident memory lymphocytes. Optimal protection in mice requires re-exposure to pneumococcus within days of initial infection. Serial surface marker phenotyping of B cell populations in a model of pneumococcal heterotypic immunity revealed that bacterial re-exposure stimulates the immediate accumulation of dynamic and heterogeneous populations of B cells in the lung, and is essential for the establishment of lung resident memory B (BRM) cells. The B cells in the early wave were activated, proliferating locally, and associated with both CD4+ T cells and CXCL13. Antagonist- and antibody-mediated interventions were implemented during this early timeframe to demonstrate that lymphocyte recirculation, CD4+ cells, and CD40 ligand (CD40L) signaling were all needed for lung BRM cell establishment, whereas CXCL13 signaling was not. While most prominent as aggregates in the loose connective tissue of bronchovascular bundles, morphometry and live lung imaging analyses showed that lung BRM cells were equally numerous as single cells dispersed throughout the alveolar septae. We propose that CD40L signaling from antigen-stimulated CD4+ T cells in the infected lung is critical to establishment of local BRM cells, which subsequently protect the airways and parenchyma against future potential infections.

Characteristics of acupoint effects on inflammation and neuro-immune interaction

A large number of studies have confirmed the anti-inflammatory effects of acupuncture, and some of the mechanisms and pathways regulating inflammatory response have been revealed. However, most of these researches focused on the effect of acupuncture on systemic anti-inflammation, and there is no consensus about the effect characteristics of different acupoints on regulating inflammatory response. It is noteworthy that increasing attention and exploration have been paid to the neuro-immune interactions and regulation of immune-inflammatory homeostasis. Importantly, the understanding of local neuroimmune regulation of non-immune organs has been deepening, which was known as the regional immunity. This new concept lays a scientific foundation for elucidating the characteristics of acupoints on the inflammation, especially the modulation of target visceral organs by the relevant acupoint stimulation. In this paper, the local effects (e.g. activating regional nerve components to induce local neuroimmuno-inflammatory regulation, etc), target visceral organ effects (e.g. regulating activities of visceral resident immune cells to initiate regional immunity regulation mediated by locally resident lymphocytes to promote inflammatory response degradation and to restore the homeostasis of regional immunity in the internal organs, via somato-visceral neuro-segmental connection, etc.) and systemic anti-inflammatory effects (e.g. regulating cholinergic anti-inflammatory pathway, including activating the vagus nerve to exert systemic anti-inflammatory effects through neuroimmune regulatory network, etc.) of acupoint stimulation were analyzed from different levels of neuroimmunological regulation, so as to provide new insights for clarifying the role of acupoints in improving inflammatory diseases.

Is Food-Triggered Atopic Dermatitis a Form of Systemic Contact Dermatitis?

Food allergy in atopic dermatitis is mediated by complex immune interactions between genetics, diet, environment, and the microbiome. When contact between inflamed skin and food antigens occurs, contact hypersensitivity can develop. Consequently, systemic contact dermatitis (SCD) can occur after ingestion of allergenic foods or food additives in the setting of a Th2 response with CLA-positive T cells, triggering dermatitis where skin resident memory lymphocytes reside. This phenomenon explains food-triggered dermatitis. Atopy patch tests (APTs) detect sensitization to food proteins responsible for SCD, which in turn can be confirmed by oral food challenge with delayed interpretation. We summarize the literature on using APTs to identify foods for oral challenge with dermatitis as an outcome. In dermatitis patients at risk for Th2 skewing based on a history of childhood-onset flexural dermatitis, shared decision-making should include a discussion of identifying and avoiding food and food additive triggers, as well as identifying and avoiding all contact allergens, prior to initiation of systemic therapy for dermatitis.

Kinetic properties of E-NTPDase activity in lymphocytes isolated from bone marrow, thymus and mesenteric lymph nodes of Wistar rats.

Plasma membrane anchored nucleotidases (E-ATPDases), as the E-NTPDase family, could hydrolyze and regulate the pericellular levels of nucleotides in lymphocytes. Each immune organ has a different microenvironment and display lymphocytes with different functions and phenotypes. Considering the different functions of each resident subpopulations of lymphocytes, the E-ATPDases activities in bone marrow (BML), thymus (TL) and mesenteric lymph node (MLL) lymphocytes of Wistar rats were characterized. The hydrolysis of extracellular nucleotides (eATP and eADP) showed linearity in time of reaction between 0 and 120min, and concentration of lymphocytes expressed in proteins between 1 and 6μg protein in the reaction medium. The optimal activity was attained at 37°C in a pH value of 8.0. The necessity of the cofactors Ca2+ and Mg2+ for the enzymatic activity was confirmed through a curve of concentration of 0-1000µM in the reaction medium, with both cofactors showing similar effects in the enzymatic activity. The Chevillard plot revealed that the hydrolysis of eATP and eADP occurred at the same active site of the enzyme. The analyses of E-ATPDases inhibitor and enzyme specificity showed possible involvement of E-NTPDase isoforms -1 and -2 in the isolated cells. Furthermore, different kinetic behavior of the nucleotide hydrolysis in each resident subpopulation lymphocyte was observed in this study, as MLL showed the higher Vmax with the lowest km values, while TL had the lowest Vmax and high km values. The kinetic values for E-NTPDase activity of each immune tissue lymphocytes can be an important therapeutic target for numeral immune-related diseases.

The multifaceted role of macrophages during acute liver injury.

The liver is situated at the interface of the gut and circulation where it acts as a filter for blood-borne and gut-derived microbes and biological molecules, promoting tolerance of non-invasive antigens while driving immune responses against pathogenic ones. Liver resident immune cells such as Kupffer cells (KCs), a subset of macrophages, maintain homeostasis under physiological conditions. However, upon liver injury, these cells and others recruited from circulation participate in the response to injury and the repair of tissue damage. Such response is thus spatially and temporally regulated and implicates interconnected cells of immune and non-immune nature. This review will describe the hepatic immune environment during acute liver injury and the subsequent wound healing process. In its early stages, the wound healing immune response involves a necroinflammatory process characterized by partial depletion of resident KCs and lymphocytes and a significant infiltration of myeloid cells including monocyte-derived macrophages (MoMFs) complemented by a wave of pro-inflammatory mediators. The subsequent repair stage includes restoring KCs, initiating angiogenesis, renewing extracellular matrix and enhancing proliferation/activation of resident parenchymal and mesenchymal cells. This review will focus on the multifaceted role of hepatic macrophages, including KCs and MoMFs, and their spatial distribution and roles during acute liver injury.

The miR-183/96/182 cluster is a checkpoint for resident immune cells and shapes the cellular landscape of the cornea

PurposeThe conserved miR-183/96/182 cluster (miR-183C) regulates both corneal sensory innervation and corneal resident immune cells (CRICs). This study is to uncover its role in CRICs and in shaping the corneal cellular landscape at a single-cell (sc) level. MethodsCorneas of naïve, young adult [2 and 6 months old (mo)], female miR-183C knockout (KO) mice and wild-type (WT) littermates were harvested and dissociated into single cells. Dead cells were removed using a Dead Cell Removal kit. CD45+ CRICs were enriched by Magnetic Activated Cell Sorting (MACS). scRNA libraries were constructed and sequenced followed by comprehensive bioinformatic analyses. ResultsThe composition of major cell types of the cornea stays relatively stable in WT mice from 2 to 6 mo, however the compositions of subtypes of corneal cells shift with age. Inactivation of miR-183C disrupts the stability of the major cell-type composition and age-related transcriptomic shifts of subtypes of corneal cells. The diversity of CRICs is enhanced with age. Naïve mouse cornea contains previously-unrecognized resident fibrocytes and neutrophils. Resident macrophages (ResMφ) adopt cornea-specific function by expressing abundant extracellular matrix (ECM) and ECM organization-related genes. Naïve cornea is endowed with partially-differentiated proliferative ResMφ and contains microglia-like Mφ. Resident lymphocytes, including innate lymphoid cells (ILCs), NKT and γδT cells, are the major source of innate IL-17a. miR-183C limits the diversity and polarity of ResMφ. ConclusionmiR-183C serves as a checkpoint for CRICs and imposes a global regulation of the cellular landscape of the cornea.

Hhip hits the brakes on lung tissue resident lymphocytes

Tissue-immune cell interactions in the periphery are well appreciated but incompletely understood. In this issue of Immunity, Wang etal. demonstrate how the disruption of a lung stromal niche leads to expansion of lung-resident lymphocytes and IFN-γ-driven exacerbation of emphysema.

Dysregulated lung stroma drives emphysema exacerbation by potentiating resident lymphocytes to suppress an epithelial stem cell reservoir

Aberrant tissue-immune interactions are the hallmark of diverse chronic lung diseases. Here, we sought to define these interactions in emphysema, a progressive disease characterized by infectious exacerbations and loss of alveolar epithelium. Single-cell analysis of human emphysema lungs revealed the expansion of tissue-resident lymphocytes (TRLs). Murine studies identified a stromal niche for TRLs that expresses Hhip, a disease-variant gene downregulated in emphysema. Stromal-specific deletion of Hhip induced the topographic expansion of TRLs in the lung that was mediated by a hyperactive hedgehog-IL-7 axis. 3D immune-stem cell organoids and animal models of viral exacerbations demonstrated that expanded TRLs suppressed alveolar stem cell growth through interferon gamma (IFNγ). Finally, we uncovered an IFNγ-sensitive subset of human alveolar stem cells that was preferentially lost in emphysema. Thus, we delineate a stromal-lymphocyte-epithelial stem cell axis in the lung that is modified by a disease-variant gene and confers host susceptibility to emphysema.

The lymphatic endothelium-derived follistatin: activin A axis regulates neutrophil motility in response to Pseudomonas aeruginosa.

The lymphatic system plays an active role during infection, however the role of lymphatic-neutrophil interactions in host-defense responses is not well understood. During infection with pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus and Yersinia pestis, neutrophils traffic from sites of infection through the lymphatic vasculature, to draining lymph nodes to interact with resident lymphocytes. This process is poorly understood, in part, due to the lack of in vitro models of the lymphatic system. Here we use a 3D microscale lymphatic vessel model to examine neutrophil-lymphatic cell interactions during host defense responses to pathogens. In previous work, we have shown that follistatin is secreted at high concentrations by lymphatic endothelial cells during inflammation. Follistatin inhibits activin A, a member of the TGF-β superfamily, and, together, these molecules form a signaling pathway that plays a role in regulating both innate and adaptive immune responses. Although follistatin and activin A are constitutively produced in the pituitary, gonads and skin, their major source in the serum and their effects on neutrophils are poorly understood. Here we report a microfluidic model that includes both blood and lymphatic endothelial vessels, and neutrophils to investigate neutrophil-lymphatic trafficking during infection with P. aeruginosa. We found that lymphatic endothelial cells produce secreted factors that increase neutrophil migration toward P. aeruginosa, and are a significant source of both follistatin and activin A during Pseudomonas infection. We determined that follistatin produced by lymphatic endothelial cells inhibits activin A, resulting in increased neutrophil migration. These data suggest that the follistatin:activin A ratio influences neutrophil trafficking during infection with higher ratios increasing neutrophil migration.

Dysregulation of CD177+ neutrophils on intraepithelial lymphocytes exacerbates gut inflammation via decreasing microbiota-derived DMF

ABSTRACT Neutrophils synergize with intestinal resident intraepithelial lymphocytes (IELs) to serve as the first-line defense and maintain intestinal homeostasis. However, the underlying mechanisms whereby neutrophils regulate IELs to inhibit intestinal inflammation are still not completely understood. Here, we found that depletion of neutrophils (especially CD177+ subset) caused expansion of colitogenic TCRγδ+CD8αα+ IELs, increased intestinal inflammation, and dysbiosis after dextran sulfate sodium exposure or Citrobacter rodentium infection in mice. scRNA-seq analysis revealed a pyroptosis-related gene signature and hyperresponsiveness to microbiota in TCRγδ+CD8αα+ IELs from colitic Cd177 −/− mice. Microbiota-derived fumarate and its derivative dimethyl fumarate (DMF), as well as fumarate-producing microbiotas, decreased in the feces of colitic Cd177 −/− mice. Elimination of dysbiosis by antibiotics treatment or co-housing procedure and DMF supplementation restrained TCRγδ+CD8αα+ IEL activation. Consistently, DMF significantly alleviated intestinal mucosal inflammation in mice through restricting gasdermin D (GSDMD)-induced pyroptosis of TCRγδ+CD8αα+ IELs. Therefore, our data reveal that neutrophils inhibit intestinal inflammation by promoting microbiota-derived DMF to regulate TCRγδ+CD8αα+ IEL activation in a GSDMD-mediated pyroptosis-dependent manner, and that DMF may serve as a therapeutic target for the management of intestinal inflammation.

Damage-induced NAD release activates intestinal CD4+ and CD8+ T cell via P2X7R signaling

BackgroundPostoperative ileus (POI) is characterized by the activation of inflammation triggered by tissue damage. Damage-associated molecular patterns (DAMPs) reportedly induce local inflammation after injury. However, the impact of DAMPs on intestinal resident lymphocytes during POI remains poorly elucidated. MethodsPOI in mice was induced via intestinal manipulation (IM). The concentration of nicotinamide adenine dinucleotide (NAD) was detected after IM. The gastrointestinal motility of the mice was assessed after IM or NAD injection. Cytokine production and calcium influx in T cells were investigated after NAD stimulation using flow cytometry. ResultsThe concentration of extracellular NAD significantly increased after IM administration, and NAD directly impaired gastrointestinal motility. Intraperitoneal injection of NAD promoted the expression of TNF-α in intestinal CD8+ and CD4+ T cells, but only IFN-γ production by CD8+ T cells was significantly promoted by NAD injection. Granzyme B production in CD8+ and CD4+ T cells decreased after administration. Concordantly, the same results were observed in NAD stimulation of intestinal CD3+ T cells in vitro. Blocking the P2X7R-related membrane enzyme ART2.2 significantly diminished the pro-inflammatory effect of NAD. ConclusionIM includes the release of NAD derived from damaged tissues, consequently promoting pro-inflammatory cytokine production in intestinal CD4+ and CD8+ T lymphocytes. NAD-induced intestinal T cells activation may be associated with POI progression in the mouse.

FLASH Modulation of Immune Microenvironment in Diffuse Midline Glioma (DMG) at Single-Cell Resolution

<h3>Purpose/Objective(s)</h3> Diffuse Midline Glioma with H3K27M mutation (DMG) is a universally fatal tumor primarily treated with radiotherapy (RT) and typically unresponsive to immune-based therapies. FLASH is a novel RT technique associated with decreased toxicity and effective tumor control in glioma models as well as immune-sparing properties in other cancer models. However, the effect of FLASH on the DMG tumor immune microenvironment (TIME) has not yet been explored. <h3>Materials/Methods</h3> Using a syngeneic murine brainstem DMG, we performed single-cell RNA sequencing on CD45+ cells isolated from tumors 4 days after posterior fossa irradiation (15Gy x 1, 9MeV), utilizing either ultra-high dose rate (90Gy/sec, FLASH) or conventional (2Gy/min, CONV) RT, and compared to unirradiated tumor (SHAM). For differential gene expression analysis, we compared CONV to SHAM (CvS) and FLASH to SHAM (FvS). We also performed single cell gene set enrichment scores (ssGSEA) for evaluation of immune signatures from gene ontology (GO); ssGSEA scores were deemed significant by a p<0.05 using Mann-Whitney U test. We validated our most robust immune markers with dual immunofluorescence. <h3>Results</h3> Unsupervised clustering of 23,165 CD45+ cells revealed 16 immune clusters, including subtypes of resident microglia (MG, 78.2% of total cells), lymphocytes (6.4%), macrophages (MPs, 5.2%), monocytes (1.8%), and dendritic cells (DCs, 1.7%). MG consisted of four distinct subtypes representing a spectrum of homeostatic to activated cellular states. In the most activated MG subtype, both CONV and FLASH showed an upregulation of interferon (IFN) genes such as Isg15 (Fold Change (FC) 1.03 CvS, 0.94 FvS) and Ifit3 (FC 0.77 CvS, 0.66 FvS) when compared to SHAM. Furthermore, ssGSEA of MG found IFN scores increased in both FvS (p < 0.0001) and CvS (p < 0.0001) while TGFbR signaling score was increased in FvS (p < 0.0001) and decreased in CvS (p < 0.0001). In non-resident myeloid clusters, the most differential response was found in MPs and DCs with robust upregulation of IFN genes for CvS (Isg15 FC 2.30 MPs, 2.67 DCs; Ifit3 FC 2.02 MPs, 2.06 DCs), while this response was not seen with FvS. This was confirmed with ssGSEA with increased IFN scores in both MPs (CvS p<0.0001, p=0.04) and DCs (CvS p<0.0001, FvS p=0.69). FLASH showed an increase in anti-inflammatory MP markers such as Mrc1, Cd163, and Maf (FC 1.40, 1.11, 1.19) and an upregulated myeloid-derived suppressor cell signature in monocytes (FvS, p < 0.0001), not seen in CvS (p = 0.09). <h3>Conclusion</h3> Our work is the first to map CONV and FLASH immune alterations with single-cell resolution in the DMG TIME. FLASH induces IFN differentially across microglia and non-resident myeloid subsets. Further exploration is warranted to understand how FLASH impacts tumor growth through differential cytokine signaling, inflammatory responses and immune checkpoint expression within the DMG TIME.

Immune defenses of the mammary gland epithelium of dairy ruminants.

The epithelium of the mammary gland (MG) fulfills three major functions: nutrition of progeny, transfer of immunity from mother to newborn, and its own defense against infection. The defense function of the epithelium requires the cooperation of mammary epithelial cells (MECs) with intraepithelial leucocytes, macrophages, DCs, and resident lymphocytes. The MG is characterized by the secretion of a large amount of a nutrient liquid in which certain bacteria can proliferate and reach a considerable bacterial load, which has conditioned how the udder reacts against bacterial invasions. This review presents how the mammary epithelium perceives bacteria, and how it responds to the main bacterial genera associated with mastitis. MECs are able to detect the presence of actively multiplying bacteria in the lumen of the gland: they express pattern recognition receptors (PRRs) that recognize microbe-associated molecular patterns (MAMPs) released by the growing bacteria. Interactions with intraepithelial leucocytes fine-tune MECs responses. Following the onset of inflammation, new interactions are established with lymphocytes and neutrophils recruited from the blood. The mammary epithelium also identifies and responds to antigens, which supposes an antigen-presenting capacity. Its responses can be manipulated with drugs, plant extracts, probiotics, and immune modifiers, in order to increase its defense capacities or reduce the damage related to inflammation. Numerous studies have established that the mammary epithelium is a genuine effector of both innate and adaptive immunity. However, knowledge gaps remain and newly available tools offer the prospect of exciting research to unravel and exploit the multiple capacities of this particular epithelium.

Distinct spatial distribution and roles of Kupffer cells and monocyte-derived macrophages in mouse acute liver injury.

Macrophages are key regulators of inflammation and repair, but their heterogeneity and multiple roles in the liver are not fully understood. We aimed herein to map the intrahepatic macrophage populations and their function(s) during acute liver injury. We used flow cytometry, gene expression analysis, multiplex-immunofluorescence, 3D-reconstruction, and spatial image analysis to characterize the intrahepatic immune landscape in mice post-CCl4-induced acute liver injury during three distinct phases: necroinflammation, and early and late repair. We observed hepatocellular necrosis and a reduction in liver resident lymphocytes during necroinflammation accompanied by the infiltration of circulating myeloid cells and upregulation of inflammatory cytokines. These parameters returned to baseline levels during the repair phase while pro-repair chemokines were upregulated. We identified resident CLEC4F+ Kupffer cells (KCs) and infiltrating IBA1+CLEC4F- monocyte-derived macrophages (MoMFs) as the main hepatic macrophage populations during this response to injury. While occupying most of the necrotic area, KCs and MoMFs exhibited distinctive kinetics, distribution and morphology at the site of injury. The necroinflammation phase was characterized by low levels of KCs and a remarkable invasion of MoMFs suggesting their potential role in phagoctosing necrotic hepatocytes, while opposite kinetics/distribution were observed during repair. During the early repair phase, yolksac - derived KCs were restored, whereas MoMFs diminished gradually then dissipated during late repair. MoMFs interacted with hepatic stellate cells during the necroinflammatory and early repair phases, potentially modulating their activation state and influencing their fibrogenic and pro-repair functions that are critical for wound healing. Altogether, our study reveals novel and distinct spatial and temporal distribution of KCs and MoMFs and provides insights into their complementary roles during acute liver injury.

LBA6 Neoadjvuant versus adjuvant pembrolizumab for resected stage III-IV melanoma (SWOG S1801)

Adjuvant therapy (AT) is currently considered for resected stage IIB-III melanoma and selected patients with resected stage IV melanoma. AT for melanoma is anti-PD-1 or targeted therapy in the presence of a BRAF mutation. Neoadjuvant therapy (NAT) with anti-PD-1 therapy is hypothesized to generate a stronger immune response from the activation of resident tumor-infiltrating lymphocytes against in vivo tumor antigens, but this has not been demonstrated in a prospective randomized trial.

Abstract 2851: Moving on: Embracing γδ T-cell biology to create truly next generation immunotherapy concepts

Abstract Chimeric antigen receptor (CAR) modification of αβ T cells has revolutionized the field of oncology, driving the focus of attention to the immune system to target and fight malignancies. Currently available αβ T-cell therapies come with many challenges, including alloreactivity, off-target toxicities, cytokine release syndrome, limited survival of infused cells and the necessity to gene edit cells to overcome graft vs. host disease. Whilst some of these limitations can be overcome with complex and costly gene-engineering approaches, utilizing innate immune cells which are inherently non-HMC restricted and able to orchestrate wider immune responses might provide an alternative strategy avoiding many of these obstacles. Many groups developed protocols to grow and modify natural killer cells and invariant natural killer T-cells whilst others, including ours, focused on the development of Vδ1 γδ T-cell-based immunotherapies. Currently, these cell types are evaluated in the clinic in both non-engineered and engineered versions. Most of these engineering principles have been directly translated from the αβ CAR-T field without taking into consideration the biology of innate immune cells: they utilize αβ T-cell specific co-stimulatory molecules and signaling cassettes as well as armoring strategies developed for αβ T-cells. Vδ1 T-cells are tissue resident lymphocytes, which for most of their lifetime remain in epithelia rich tissues. They consistently survey tissues and monitor malignant transformation using a variety of natural cytotoxicity receptors, NK like receptors and the γδ TCR. In contrast to αβ T-cells, Vδ1 T-cells do not follow the ‘two-signal theory’: they must integrate multiple signals from an array of receptors in order to discriminate between healthy and malignant cells. We thus propose that CAR strategies exclusively utilizing CD3ζ activation domains are not using the full potential of innate immune cells for cellular immunotherapy. Another distinguishing feature of Vδ1 T-cells is the absence of traditional autocrine cytokine feedback loops seen in αβ T-cells. Our results demonstrate that traditional cytokine armoring strategies utilizing forced secretion of soluble or membrane-tethered IL-15 are detrimental to the biology of Vδ1 T-cells. By dissecting the IL-15 receptor biology we are now able to create cells that not only survive and grow in the absence of exogenous IL-15 but become more sensitive to endogenous levels. Applying γδ T-cell biology and an adapted understanding of the IL-15 pathway, we have now created novel engineering strategies to tailor specificity, potency, and proliferation of Vδ1 T-cells even in the presence of CAR whilst maintaining the cells’ inherent ability to discriminate between healthy and malignant cells. Preclinical evaluation of these concepts is ongoing with the aim to develop next generation tailored Vδ1T-cell immunotherapies. Citation Format: Jyothi Kumaran, Rajeev Karattil, André Simoes, Rebecca Alade, Mihil Patel, Liz Wood, Gonzalo Mercado Vico, Amy Lane, Sara Tamagno, Sarah Edwards, Andrea Venuso, Sam Illingworth, Alice Brown, Michael Koslowski, Istvan Kovacs, Oliver Nussbaumer. Moving on: Embracing γδ T-cell biology to create truly next generation immunotherapy concepts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2851.