Tissue-resident regulatory T cells accumulate at human barrier lymphoid organs.

Regulatory T cells (Tregs) play a critical role in immune regulation and peripheral tolerance. While different types of Tregs have been identified in both mice and humans, much of our understanding about how these cells maintain immune homeostasis is derived from animal models. In this study, we examined two distinct human lymphoid organs to understand how repeated exposure to infections at the mucosal surface influences the phenotype and tissue localization of Tregs. We show that while Tregs in both tonsils and spleen express a tissue-resident phenotype, they accumulate in greater numbers in tonsils. Tonsillar-resident Tregs exhibit a highly suppressive phenotype with significantly increased expression of CD39, ICOS and CTLA-4 compared with their counterparts in circulation or in the spleen. Functionally, resident Tregs are able effectively to suppress T cell proliferation. We further demonstrate that tonsillar-resident Tregs share key features of T follicular helper cells. Spatial analysis reveals that the vast majority of resident Tregs are localized at the border of the T-zone and B cell follicle, as well as within the lymphocyte pockets enriched with resident memory T cells. Together our findings suggest that resident Tregs are strategically co-localized to maintain immune homeostasis at sites of recurrent inflammation.

A proinflammatory CD4+ T cell phenotype in gestational diabetes mellitus.

AIMS/HYPOTHESIS: Numerous adaptations of the maternal immune system are necessary during pregnancy to maintain immunological tolerance to the semi-allogeneic fetus. Several complications of pregnancy have been associated with dysregulation of these adaptive mechanisms. While gestational diabetes mellitus (GDM) has been associated with upregulation of circulating inflammatory factors linked to innate immunity, polarisation of the adaptive immune system has not been extensively characterised in this condition. We aimed to characterise pro- and anti-inflammatory CD4+ (T helper [Th]) T cell subsets in women with GDM vs women without GDM (of similar BMI), during and after pregnancy, and examine the relationship between CD4+ subsets and severity of GDM. METHODS: This is a prospective longitudinal case-control study of 55 women with GDM (cases) and 65 women without GDM (controls) at a tertiary maternity hospital. Quantification of proinflammatory (Th17, Th17.1, Th1) and anti-inflammatory (regulatory T cell [Treg]) CD4+ T cell subsets was performed on peripheral blood at 37 weeks gestation and 7 weeks postpartum, and correlated with clinical characteristics and measures of blood glucose. RESULTS: Women with GDM had a significantly greater percentage of Th17 (median 2.49% [interquartile range 1.62-4.60] vs 1.85% [1.13-2.98], p = 0.012) and Th17.1 (3.06% [1.30-4.33] vs 1.55% [0.65-3.13], p = 0.006) cells compared with the control group of women without GDM. Women with GDM also had higher proinflammatory cell ratios (Th17:Treg, Th17.1:Treg and Th1:Treg) in pregnancy compared with the control group of women without GDM. In the control group, there was a statistically significant independent association between 1 h glucose levels in the GTT and Th17 cell percentages, and also between 2 h glucose levels and percentage of Th17 cells. The percentage of Th17 cells and the Th17:Treg ratio declined significantly after delivery in women with GDM, whereas this was not the case with the control group of women. Nevertheless, a milder inflammatory phenotype persisted after delivery (higher Th17:Treg ratio) in women with GDM vs women without. CONCLUSIONS/INTERPRETATION: Dysregulation of adaptive immunity supports a novel paradigm of GDM that extends beyond hyperglycaemia and altered innate immunity.

Medium-term assessment of cardiac function in pediatric cancer survivors. Comparison of different echocardiographic methods, cardiac MRI and cardiac biomarker testing in adolescent cancer survivors.

PURPOSE: To evaluate the feasibility and correlation of 3D echocardiography (echo) and cardiac biomarkers with cardiac MRI, in surveillance of cardiac function for cancer survivors. METHODS: Subjects ≥10 years of age who have survived >2 years after completion of cancer treatment from a single center were enrolled. Cardiac MRI and 3D echo images were obtained on the same day when routine echo was obtained. On the same day, along with annual routine blood test, cardiac biomarkers N-terminal pro-B-type natriuretic peptide levels (NT-proBNP) and troponin-I levels were also measured. RESULTS: Cardiac MRI was feasible in all 50 subjects. Three-dimensional echo and 2D echo images were of poor quality in four subjects. With a median duration of remission of 10 years, there were four subjects with mild LV dysfunction (cardiac MRI LV EF of<53%). None had MRI EF <50%, and nine subjects had LVEF <55%. M-mode echo overestimated EF more than 2D and 3D echo. Two-dimensional and 3D echo methods had much tighter limits of agreement for LV EF. For measurement of LVEF, 3D echo had a lower % error than 2D echo or M-mode echo. One subject had an abnormal troponin-I level and another one had an elevated NT-proBNP. CONCLUSIONS: Three-dimensional echo can be performed in most adolescent cancer survivors, and it correlates well with MRI. Further large-scale research is required in assessing utility of cardiac biomarkers in pediatric cancer survivors.

Association of Biotin Ingestion With Performance of Hormone and Nonhormone Assays in Healthy Adults.

Importance: Biotinylated antibodies and analogues, with their strong binding to streptavidin, are used in many clinical laboratory tests. Excess biotin in blood due to supplemental biotin ingestion may affect biotin-streptavidin binding, leading to potential clinical misinterpretation. However, the degree of interference remains undefined in healthy adults. Objective: To assess performance of specific biotinylated immunoassays after 7 days of ingesting 10 mg/d of biotin, a dose common in over-the-counter supplements for healthy adults. Design, Setting, and Participants: Nonrandomized crossover trial involving 6 healthy adults who were treated at an academic medical center research laboratory. Exposure: Administration of 10 mg/d of biotin supplementation for 7 days. Main Outcomes and Measures: Analyte concentrations were compared with baseline (day 0) measures on the seventh day of biotin treatment and 7 days after treatment had stopped (day 14). The 11 analytes included 9 hormones (ie, thyroid-stimulating hormone, total thyroxine, total triiodothyronine, free thyroxine, free triiodothyronine, parathyroid hormone, prolactin, N-terminal pro-brain natriuretic peptide, 25-hydroxyvitamin D) and 2 nonhormones (prostate-specific antigen and ferritin). A total of 37 immunoassays for the 11 analytes were evaluated on 4 diagnostic systems, including 23 assays that incorporated biotin and streptavidin components and 14 assays that did not include biotin and streptavidin components and served as negative controls. Results: Among the 2 women and 4 men (mean age, 38 years [range, 31-45 years]) who took 10 mg/d of biotin for 7 days, biotin ingestion-associated interference was found in 9 of the 23 (39%) biotinylated assays compared with none of the 14 nonbiotinylated assays (P = .007). Results from 5 of 8 biotinylated (63%) competitive immunoassays tested falsely high and results from 4 out of 15 (27%) biotinylated sandwich immunoassays tested falsely low. Conclusions and Relevance: In this preliminary study of 6 healthy adult participants and 11 hormone and nonhormone analytes measured by 37 immunoassays, ingesting 10 mg/d of biotin for 1 week was associated with potentially clinically important assay interference in some but not all biotinylated assays studied. These findings should be considered for patients taking biotin supplements before ordering blood tests or when interpreting results. Trial Registration: clinicaltrials.gov Identifier: NCT03034707.

It's Complicated: Navigating Multiple Identities in Small Town America.

During the past few years, research focused on individuals who identify as gay, lesbian, bisexual, and transgender (LGBT) have increased. However, much of this literature focuses on White LGBT individuals, and less on individuals who have intersected identities, such as being both gay/lesbian and a person of color. Consequently, this population becomes obscured in scholarly discourses, thus being made "invisible" with regard to theory, research, and clinical interventions, with the result that clinicians are provided little or no knowledge of theories or best practices when working with individuals who are both sexual minority and people of color. This article discusses an integrated theoretical and treatment process when working with clients who are navigating multiple oppressed identities.

Hemagglutinin-neuraminidase balance confers respiratory-droplet transmissibility of the pandemic H1N1 influenza virus in ferrets.

A novel reassortant derived from North American triple-reassortant (TRsw) and Eurasian swine (EAsw) influenza viruses acquired sustained human-to-human transmissibility and caused the 2009 influenza pandemic. To identify molecular determinants that allowed efficient transmission of the pandemic H1N1 virus among humans, we evaluated the direct-contact and respiratory-droplet transmissibility in ferrets of representative swine influenza viruses of different lineages obtained through a 13-y surveillance program in southern China. Whereas all viruses studied were transmitted by direct contact with varying efficiency, respiratory-droplet transmissibility (albeit inefficient) was observed only in the TRsw-like A/swine/Hong Kong/915/04 (sw915) (H1N2) virus. The sw915 virus had acquired the M gene derived from EAsw and differed from the gene constellation of the pandemic H1N1 virus by the neuraminidase (NA) gene alone. Glycan array analysis showed that pandemic H1N1 virus A/HK/415742/09 (HK415742) and sw915 possess similar receptor-binding specificity and affinity for α2,6-linked sialosides. Sw915 titers in differentiated normal human bronchial epithelial cells and in ferret nasal washes were lower than those of HK415742. Introducing the NA from pandemic HK415742 into sw915 did not increase viral replication efficiency but increased respiratory-droplet transmissibility, despite a substantial amino acid difference between the two viruses. The NA of the pandemic HK415742 virus possessed significantly higher enzyme activity than that of sw915 or other swine influenza viruses. Our results suggest that a unique gene constellation and hemagglutinin-neuraminidase balance play a critical role in acquisition of efficient and sustained human-to-human transmissibility.

Cutaneous malignancies in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a common lymphoid malignancy that is associated with an increased risk of developing cutaneous malignancies. Clinical outcomes for these malignancies, including melanoma and keratinocyte cancers (KC), are worse for patients with CLL. Individuals with CLL develop an immunodeficiency of both the adaptive and innate immune system, which plays a role in the increased prevalence of skin cancers. This review focuses on the complex interplay between genetics, immunity, and pathogens that influence the cellular composition and biology of skin tumors and their microenvironment in CLL patients, and in comparison with other chronic hematological malignancies. It is paramount for dermatologists to be aware of the association between CLL (and chronic hematological malignancies more broadly) and cutaneous malignancies. This is a high-risk population who require regular and vigorous dermatologic follow-up.

Emergent trees in Colophospermum mopane woodland: influence of elephant density on persistence versus attrition.

Colophospermum mopane (mopane) forms mono-dominant woodlands covering extensive areas of southern Africa. Mopane provides a staple foodstuff for elephants, who hedge woodland by reducing trees to small trees or shrubs, leaving emergent trees which are too large to be pollarded. Emergent trees are important for supporting faunal biodiversity, but they can be killed by ringbarking. This study first examined the influence of elephant density on woodland transformation and the height distribution of canopy volume, and, second, whether canopy volume is maintained, and tall emergent trees too large to be broken can persist, under chronic elephant utilisation. Three regimes of 0.23, 0.59 and 2.75 elephants km-2 differed in vegetation structure and the height structure of trees. Areas under the highest elephant density supported the lowest total canopy volume owing to less canopy for plants >3 m in height, shorter trees, loss of most trees 6-10 m in height, but trees >10 m in height (>45 cm stem diameter) persisted. Under eight years of chronic utilisation by elephants, transformed mopane woodland maintained its plant density and canopy volume. Plant density was greatest for the 0-1 m height class, whereas the 3.1-6 m height class provided the bulk of canopy volume, and the 1.1-3 m height layer contained the most canopy volume. Emergent trees (>10 m in height) suffered a loss of 1.4% per annum as a result of debarking. Canopy dieback of emergent trees increased conspicuously when more than 50% of a stem was debarked, and such trees could be toppled by windthrow before being ringbarked. Thus relict emergent trees will slowly be eliminated but will not be replaced whilst smaller trees are being maintained in a pollarded state. Woodland transformation has not markedly reduced canopy volume available to elephants, but the slow attrition of emergent trees may affect supported biota, especially cavity-dependent vertebrate species, making use of these trees.

Corrigendum: Late-stage MC38 tumours recapitulate features of human colorectal cancer - implications for appropriate timepoint selection in preclinical studies.

[This corrects the article DOI: 10.3389/fimmu.2023.1152035.].

CD4+ T cell immunity against cutaneous melanoma encompasses multifaceted MHC II-dependent responses.

Whereas CD4+ T cells conventionally mediate antitumor immunity by providing help to CD8+ T cells, recent clinical studies have implied an important role for cytotoxic CD4+ T cells in cancer immunity. Using an orthotopic melanoma model, we provide a detailed account of antitumoral CD4+ T cell responses and their regulation by major histocompatibility complex class II (MHC II) in the skin. Intravital imaging revealed prominent interactions of CD4+ T cells with tumor debris-laden MHC II+ host antigen-presenting cells that accumulated around tumor cell nests, although direct recognition of MHC II+ melanoma cells alone could also promote CD4+ T cell control. CD4+ T cells stably suppressed or eradicated tumors even in the absence of other lymphocytes by using tumor necrosis factor-α and Fas ligand (FasL) but not perforin-mediated cytotoxicity. Interferon-γ was critical for protection, acting both directly on melanoma cells and via induction of nitric oxide synthase in myeloid cells. Our results illustrate multifaceted and context-specific aspects of MHC II-dependent CD4+ T cell immunity against cutaneous melanoma, emphasizing modulation of this axis as a potential avenue for immunotherapies.

A simple isotope dilution electrospray ionization tandem mass spectrometry method for the determination of free phenytoin.

BACKGROUND: Phenytoin (diphenylhydantoin) is an anticonvulsant drug frequently prescribed for the treatment of many types of seizures. Because the drug is highly protein bound (90%-95%) and many conditions can displace the drug from proteins, the measurement of free phenytoin is warranted. Due to the unavailability of free phenytoin assays in many chemistry analyzers or limitations of immunoassays, chromatographic methods such as liquid chromatography-tandem mass spectrometry (LC-MS-MS) are preferred for the assay of free phenytoin. METHODS: The sample preparation involved ultrafiltration of serum or plasma to separate free phenytoin. Acetonitrile containing internal standard, phenytoin-d10, was added to the ultrafiltrate. The samples were centrifuged, and supernatants were injected into an LC-MS-MS involving reverse phase Ultra BiPh 5-µm × 50 × 2.1-mm analytical column, and mobile phases, water and methanol containing 0.1% formic acid. The mass/charge (m/z) transitions were as follows: phenytoin -253.0 > 182.2 and 253.0 > 104.00; phenytoin-d10 -263.2 > 192.12. RESULTS: Linearity of the method ranged from 0.1 to 4.0 µg/mL. Within-run and between-run imprecision values were <5% and <10%, respectively. The samples were stable for 2 weeks at 4°C and 4 weeks at -20°C. The method compared well with the laborious liquid-liquid extraction method and did not show any significant ion suppression or enhancement. CONCLUSIONS: A simple LC-MS-MS method was developed for the assay of free phenytoin. The method does not require laborious liquid-liquid or solid-phase extraction. The method has high analytical sensitivity, low imprecision, and a wide analytical measurement range.

The Tumor Immune Microenvironment in Cutaneous Squamous Cell Carcinoma Arising in Organ Transplant Recipients.

Cutaneous squamous cell carcinoma (cSCC) is the most common malignancy in immune-suppressed organ transplant recipients (OTRs). Whilst rates of other malignancies (both cutaneous and non-cutaneous) are elevated in this population, the increase is far less striking. This suggests that cSCC must be a highly immunogenic tumor. The tumor immune microenvironment is altered in cSCC from OTRs. It has reduced anti-tumor properties and instead provides an environment that facilitates tumor growth and survival. Understanding the composition and function of the tumor immune microenvironment in cSCC from OTRs is useful for prognostication and therapeutic decisions.

Personal recovery associated with deep brain stimulation for treatment-resistant depression: A constructivist grounded theory study.

WHAT IS KNOWN ON THE SUBJECT?: Major depressive disorder is the most prevalent of all mental illnesses. 10%-20% of patients with depression and 1% of the population overall have treatment-resistant depression (TRD). DBS is an emerging investigational treatment for TRD with documented clinical efficacy and safety. The framework of the recovery model includes both clinical and personal recovery. Personal recovery is a self-process in which hope, empowerment and optimism are embraced to overcome the impact of mental illness on one's sense of self. Although clinical and functional outcomes of DBS for TRD have been well documented in the previous studies, personal recovery as an outcome has been explored only in a handful of studies. WHAT THIS PAPER ADDS TO EXISTING KNOWLEDGE?: This is the first qualitative study exploring personal recovery from DBS treatment specific to the target of subcallosal cingulate cortex in patients with TRD. Since the existing literature on personal recovery in DBS studies is limited, the contribution of this paper is crucial to this field. For individuals who responded to deep brain stimulation clinically, neither participants nor family believed it cured their depression, but rather there was a significant decrease in the severity of symptoms of depression. A holistic-oriented framework (that includes personal recovery) is significant for those individuals with TRD undergoing DBS. Personal and clinical recovery are two different constructs, and individuals may experience one or the other or both. The experience of participants who responded to deep brain stimulation recognized that the recovery from depression is a process of reconstructing self. This process involved a period of adjustment that evoked a deeper self-awareness, re-engagement with daily living and newfound gratitude in living. Individuals transitioned from an emotionally driven life to one where future goals were considered. Supportive relationships were instrumental in this process. WHAT ARE THE IMPLICATIONS FOR PRACTICE?: A deep brain stimulation intervention for treatment-resistant depression offered individuals an opportunity for personal recovery where a reconstruction of self occurred. Personal recovery can be considered as an outcome in conjunction with clinical and functional outcomes in future DBS trials for TRD. The relevance of personal recovery in the prevention of relapses needs further investigation. To advocate for care and services that facilitate the process of recovery from depression, it is important to understand the personal dimensions and experience of recovery that may influence the process. To develop recovery-oriented interventions to help patients and families in recovery post-deep brain stimulation, further understanding of support and negotiating relationships during this life-altering experience is needed. ABSTRACT: Introduction Multiple trials of antidepressant treatments in patients with depression pose a major challenge to the mental health system. Deep brain stimulation (DBS) is an emerging and promising investigational treatment to reduce depressive symptoms in individuals with treatment-resistant depression (TRD). The clinical and functional outcomes of DBS for TRD have been well documented in previous studies; however, studies of personal recovery as an outcome of DBS specific to the target of subcallosal cingulate cortex in patients with TRD are limited. Aim To explore the processes of personal recovery in patients with treatment-resistant depression following subcallosal cingulate-deep brain stimulation. Method Participants were 18 patients with TRD who participated in the subcallosal cingulate (SCC)-DBS trial and 11 family members. They also participated in add-on individual cognitive behavioural therapy during the trial. A qualitative constructivist grounded theory approach was used to conceptualize the personal recovery process of patients and families. Results While every participant and their families' journey were unique following the deep brain stimulation intervention, a theoretical model of Balancing to Establish a Reconstructed Self emerged from the data. The themes underlying the model were (1) Balancing to Establish a Reconstructed Self: A Whole-Body Experience, (2) The Liminal Space in-between: Balancing with Cautious Optimism, (3) Hope: Transitioning from Emotion-Focussed Living to Goal-Oriented Planning and (4) Support: Negotiating Relationships. Discussion This is the first study examining recovery from patients' perspectives as an outcome of SCC-DBS intervention for TRD. The study shows that personal recovery is a gradual and continual process of reconstruction of the self, developing through supportive relationships. Clinical and personal recovery are two distinct constructs, and individuals may experience one or the other or both. Most patients who do respond clinically experience improvement in terms of having optimism and hope. Some patients, however, respond with significant symptom reduction but are not able to achieve personal recovery to experience joy or hope for improved quality of living. Implications for Practice Strategies for personal recovery for both patients and family need to be considered during and post deep brain stimulation intervention. Nurses working with these patients and families may benefit from education, training and support to assess and engage in conversations about their recovery process.

Late-stage MC38 tumours recapitulate features of human colorectal cancer - implications for appropriate timepoint selection in preclinical studies.

Anti-tumour T cell responses play a crucial role in controlling the progression of colorectal cancer (CRC), making this disease a promising candidate for immunotherapy. However, responses to immune-targeted therapies are currently limited to subpopulations of patients and specific types of cancer. Clinical studies have therefore focussed on identifying biomarkers that predict immunotherapy responses and elucidating the immunological landscapes of different cancers. Meanwhile, our understanding of how preclinical tumour models resemble human disease has fallen behind, despite their crucial role in immune-targeted drug development. A deeper understanding of these models is therefore needed to improve the development of immunotherapies and the translation of findings made in these systems. MC38 colon adenocarcinoma is a widely used preclinical model, yet how it recapitulates human colorectal cancer remains poorly defined. This study investigated the tumour-T cell immune landscape of MC38 tumours using histology, immunohistochemistry, and flow cytometry. We demonstrate that early-stage tumours exhibit a nascent TME, lacking important immune-resistance mechanisms of clinical interest, while late-stage tumours exhibit a mature TME resembling human tumours, with desmoplasia, T cell exhaustion, and T cell exclusion. Consequently, these findings clarify appropriate timepoint selection in the MC38 model when investigating both immunotherapies and mechanisms that contribute to immunotherapy resistance. Overall, this study provides a valuable resource that will enable appropriate application of the MC38 model and expedite the development and clinical translation of new immunotherapies.

A unique cytotoxic CD4+ T cell-signature defines critical COVID-19.

Objectives: SARS-CoV-2 infection causes a spectrum of clinical disease presentation, ranging from asymptomatic to fatal. While neutralising antibody (NAb) responses correlate with protection against symptomatic and severe infection, the contribution of the T-cell response to disease resolution or progression is still unclear. As newly emerging variants of concern have the capacity to partially escape NAb responses, defining the contribution of individual T-cell subsets to disease outcome is imperative to inform the development of next-generation COVID-19 vaccines. Methods: Immunophenotyping of T-cell responses in unvaccinated individuals was performed, representing the full spectrum of COVID-19 clinical presentation. Computational and manual analyses were used to identify T-cell populations associated with distinct disease states. Results: Critical SARS-CoV-2 infection was characterised by an increase in activated and cytotoxic CD4+ lymphocytes (CTL). These CD4+ CTLs were largely absent in asymptomatic to severe disease states. In contrast, non-critical COVID-19 was associated with high frequencies of naïve T cells and lack of activation marker expression. Conclusion: Highly activated and cytotoxic CD4+ T-cell responses may contribute to cell-mediated host tissue damage and progression of COVID-19. Induction of these potentially detrimental T-cell responses should be considered when developing and implementing effective COVID-19 control strategies.

Intratumoral CD16+ Macrophages Are Associated with Clinical Outcomes of Patients with Metastatic Melanoma Treated with Combination Anti-PD-1 and Anti-CTLA-4 Therapy.

PURPOSE: This study characterizes intratumoral macrophage populations within baseline melanoma biopsies from patients with advanced melanoma who received either anti-PD-1 monotherapy or a combination with anti-CTLA-4. Particularly, FcγRIIIa (CD16)-expressing macrophage densities were investigated for associations with response and progression-free survival. EXPERIMENTAL DESIGN: Patients with advanced melanoma who received either anti-PD-1 monotherapy or combination anti-PD-1 and anti-CTLA-4 were retrospectively identified. Macrophage populations were analyzed within baseline melanoma biopsies via multiplex IHC in relation to treatment outcomes. RESULTS: Patients who responded to combination immune checkpoint inhibitor contained higher CD16+ macrophage densities than those who did not respond (196 vs. 7 cells/mm2; P = 0.0041). There was no diffidence in CD16+ macrophage densities in the PD-1 monotherapy-treated patients based on response (118 vs. 89 cells/mm2; P = 0.29). A significantly longer 3-year progression-free survival was observed in combination-treated patients with high intratumoral densities of CD16+ macrophages compared with those with low densities (87% vs. 42%, P = 0.0056, n = 40). No association was observed in anti-PD-1 monotherapy-treated patients (50% vs. 47%, P = 0.4636, n = 50). Melanoma biopsies with high densities of CD16+ macrophages contained upregulated gene expression of critical T-cell recruiting chemokines (CXCL9, CXCL10, and CXCL11). CONCLUSIONS: Our data demonstrate that tumor microenvironments enriched with CD16+ macrophages are favorable for response to combination anti-PD-1 and anti-CTLA-4 therapy but not anti-PD-1 monotherapy. These data provides a potential biomarker of response for combination immunotherapies in patients with metastatic melanoma. See related commentary by Smithy and Luke, p. 2345.

Approaches to spatially resolving the tumour immune microenvironment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common and deadly cancer worldwide. Many factors contribute to mortality and place an individual at high risk of developing HCC, including viral infection, alcohol intake, metabolic-associated disease, autoimmunity and genetic liver disorders. Although there are many therapeutics available, much about this disease remains to be understood. This is most evident when investigating the tumour microenvironment (TME). Both innate and adaptive immune cells have been associated with carcinogenesis within the TME of HCC patients. The ability to interrogate the TME more thoroughly with spatial technologies continues to improve, both at the experimental and analytical stages. This review provides insight into technologies available to investigate the TME, and how such technologies are beneficial for improving our understanding of HCC carcinogenesis.

Exposure to solar ultraviolet radiation establishes a novel immune suppressive lipidome in skin-draining lymph nodes.

The ability of ultraviolet radiation to suppress the immune system is thought to be central to both its beneficial (protection from autoimmunity) and detrimental (carcinogenic) effects. Previous work revealed a key role for lipids particularly platelet-activating factor and sphingosine-1-phosphate in mediating UV-induced immune suppression. We therefore hypothesized that there may be other UV-induced lipids that have immune regulatory roles. To assess this, mice were exposed to an immune suppressive dose of solar-simulated UV (8 J/cm2). Lipidomic analysis identified 6 lipids (2 acylcarnitines, 2 neutral lipids, and 2 phospholipids) with significantly increased levels in the skin-draining lymph nodes of UV-irradiated mice. Imaging mass spectrometry of the lipids in combination with imaging mass cytometry identification of lymph node cell subsets indicated a preferential location of UV-induced lipids to T cell areas. In vitro co-culture of skin-draining lymph node lipids with lymphocytes showed that lipids derived from UV-exposed mice have no effect on T cell activation but significantly inhibited T cell proliferation, indicating that the lipids play an immune regulatory role. These studies are important first steps in identifying novel lipids that contribute to UV-mediated immune suppression.

High-Dimensional and Spatial Analysis Reveals Immune Landscape-Dependent Progression in Cutaneous Squamous Cell Carcinoma.

PURPOSE: The tumor immune microenvironment impacts the biological behavior of the tumor, but its effect on clinical outcomes in head and neck cutaneous squamous cell carcinomas (HNcSCC) is largely unknown. EXPERIMENTAL DESIGN: We compared the immune milieu of high-risk HNcSCC that never progressed to metastasis with those that metastasized using multiparameter imaging mass cytometry. The cohort included both immunosuppressed patients (IS) and patients with an absence of clinical immune-suppression (ACIS). Spatial analyses were used to identify cellular interactions that were associated with tumor behavior. RESULTS: Nonprogressing primary HNcSCC were characterized by higher CD8+ and CD4+ T-cell responses, including numerically increased regulatory T cells. In contrast, primary lesions from HNcSCC patients who progressed were largely devoid of T cells with lower numbers of innate immune cells and increased expression of checkpoint receptors and in the metastatic lesions were characterized by an accumulation of B cells. Spatial analysis reveals multiple cellular interactions associated with nonprogressing primary tumors that were distinct in primary tumors of disease-progressing patients. Cellular regional analysis of the tumor microenvironment also shows squamous cell-enriched tumor regions associated with primary nonprogressing tumors. CONCLUSIONS: Effective responses from both CD8+ and CD4+ T cells in the tumor microenvironment are essential for immune control of primary HNcSCC. Our findings indicate that the early events that shape the immune responses in primary tumors dictate progression and disease outcomes in HNcSCC.

Detailed spatial immunophenotyping of primary melanomas reveals immune cell subpopulations associated with patient outcome.

While the tumor immune microenvironment (TIME) of metastatic melanoma has been well characterized, the primary melanoma TIME is comparatively poorly understood. Additionally, although the association of tumor-infiltrating lymphocytes with primary melanoma patient outcome has been known for decades, it is not considered in the current AJCC melanoma staging system. Detailed immune phenotyping of advanced melanoma has revealed multiple immune biomarkers, including the presence of CD8+ T-cells, for predicting response to immunotherapies. However, in primary melanomas, immune biomarkers are lacking and CD8+ T-cells have yet to be extensively characterized. As recent studies combining immune features and clinicopathologic characteristics have created more accurate predictive models, this study sought to characterize the TIME of primary melanomas and identify predictors of patient outcome. We first phenotyped CD8+ T cells in fresh stage II primary melanomas using flow cytometry (n = 6), identifying a CD39+ tumor-resident CD8+ T-cell subset enriched for PD-1 expression. We then performed Opal multiplex immunohistochemistry and quantitative pathology-based immune profiling of CD8+ T-cell subsets, along with B cells, NK cells, Langerhans cells and Class I MHC expression in stage II primary melanoma specimens from patients with long-term follow-up (n = 66), comparing patients based on their recurrence status at 5 years after primary diagnosis. A CD39+CD103+PD-1- CD8+ T-cell population (P2) comprised a significantly higher proportion of intratumoral and stromal CD8+ T-cells in patients with recurrence-free survival (RFS) ≥5 years vs those with RFS <5 years (p = 0.013). Similarly, intratumoral B cells (p = 0.044) and a significantly higher B cell density at the tumor/stromal interface were associated with RFS. Both P2 and B cells localized in significantly closer proximity to melanoma cells in patients who remained recurrence-free (P2 p = 0.0139, B cell p = 0.0049). Our results highlight how characterizing the TIME in primary melanomas may provide new insights into how the complex interplay of the immune system and tumor can modify the disease outcomes. Furthermore, in the context of current clinical trials of adjuvant anti-PD-1 therapies in high-risk stage II primary melanoma, assessment of B cells and P2 could identify patients at risk of recurrence and aid in long-term treatment decisions at the point of primary melanoma diagnosis.