Immune Response In Lymph Nodes Research Articles

Orchestrating antigen delivery and presentation efficiency in lymph node by nanoparticle shape for immune response.

Activating humoral and cellular immunity in lymph nodes (LNs) of nanoparticle-based vaccines is critical to controlling tumors. However, how the physical properties of nanovaccine carriers orchestrate antigen capture, lymphatic delivery, antigen presentation and immune response in LNs is largely unclear. Here, we manufactured gold nanoparticles (AuNPs) with the same size but different shapes (cages, rods, and stars), and loaded tumor antigen as nanovaccines to explore their disparate characters on above four areas. Results revealed that star-shaped AuNPs captured and retained more repetitive antigen epitopes. On lymphatic delivery, both rods and star-shaped nanovaccines mainly drain into the LN follicles region while cage-shaped showed stronger paracortex retention. A surprising finding is that the star-shaped nanovaccines elicited potent humoral immunity, which is mediated by CD4+ T helper cell and follicle B cell cooperation significantly preventing tumor growth in the prophylactic study. Interestingly, cage-shaped nanovaccines preferentially presented peptide-MHC I complexes to evoke robust CD8+ T cell immunity and showed the strongest therapeutic efficacy when combined with the PD-1 checkpoint inhibitor in established tumor study. These results highlight the importance of nanoparticle shape on antigen delivery and presentation for immune response in LNs, and our findings support the notion that different design strategies are required for prophylactic and therapeutic vaccines.

Multiscale deep learning framework captures systemic immune features in lymph nodes predictive of triple negative breast cancer outcome in large-scale studies.

The suggestion that the systemic immune response in lymph nodes (LNs) conveys prognostic value for triple-negative breast cancer (TNBC) patients has not previously been investigated in large cohorts. We used a deep learning (DL) framework to quantify morphological features in haematoxylin and eosin-stained LNs on digitised whole slide images. From 345 breast cancer patients, 5,228 axillary LNs, cancer-free and involved, were assessed. Generalisable multiscale DL frameworks were developed to capture and quantify germinal centres (GCs) and sinuses. Cox regression proportional hazard models tested the association between smuLymphNet-captured GC and sinus quantifications and distant metastasis-free survival (DMFS). smuLymphNet achieved a Dice coefficient of 0.86 and 0.74 for capturing GCs and sinuses, respectively, and was comparable to an interpathologist Dice coefficient of 0.66 (GC) and 0.60 (sinus). smuLymphNet-captured sinuses were increased in LNs harbouring GCs (p < 0.001). smuLymphNet-captured GCs retained clinical relevance in LN-positive TNBC patients whose cancer-free LNs had on average ≥2 GCs, had longer DMFS (hazard ratio [HR] = 0.28, p = 0.02) and extended GCs' prognostic value to LN-negative TNBC patients (HR = 0.14, p = 0.002). Enlarged smuLymphNet-captured sinuses in involved LNs were associated with superior DMFS in LN-positive TNBC patients in a cohort from Guy's Hospital (multivariate HR = 0.39, p = 0.039) and with distant recurrence-free survival in 95 LN-positive TNBC patients of the Dutch-N4plus trial (HR = 0.44, p = 0.024). Heuristic scoring of subcapsular sinuses in LNs of LN-positive Tianjin TNBC patients (n = 85) cross-validated the association of enlarged sinuses with shorter DMFS (involved LNs: HR = 0.33, p = 0.029 and cancer-free LNs: HR = 0.21 p = 0.01). Morphological LN features reflective of cancer-associated responses are robustly quantifiable by smuLymphNet. Our findings further strengthen the value of assessment of LN properties beyond the detection of metastatic deposits for prognostication of TNBC patients. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract P5-01-01: Multiscale Deep Learning framework to capture systemic immune features in lymph nodes predictive of triple negative breast cancer outcome

Abstract Systemic immune responses in lymph nodes (LN) convey significant prognostic value for breast cancer patients, which can inform disease progression and optimal treatment management. However, have, so far, not been assessed in large patient cohorts. We have previously shown that morphological alterations in axillary LNs, namely the formation of germinal centres (GCs) in cancer-free LNs, add prognostic value to tumour infiltrating lymphocytes (TILs) in triple-negative breast cancer patients (TNBC) for the development of distant metastasis. Extending manual assessment of LNs beyond the detection of cancer requires the integration of robust deep learning pipelines into the digital pathology workflow. Here, we propose a supervised multiscale deep learning framework named smuLymphNet to capture and quantify GCs and sinuses within LNs from digitised Haematoxylin and Eosin-stained (H&amp;E) whole slide images (WSIs) and show good concordance compared with an inter-pathologist Dice coefficient of manual annotations from four pathologists. The smuLymphNet framework consists of (i) a detection algorithm to determine the boundaries of each LN section on the WSI, using an Otsu-based thresholding method and contouring algorithm; (ii) a supervised multiscale deep learning module for the segmentation of GCs and sinuses; and (iii) quantification of the number, size, and shape of the predicted features. We applied smuLymphNet to a total of 1,800 H&amp;E-stained WSI of &amp;gt;4,000 cancer-free and involved LNs from a retrospectively collected breast cancer cohort collected at Guy’s Hospital (London, UK) from 177 patients (122 N+) enriched for the triple-negative phenotype. A subset of 114 WSI and five breast cancer LN WSIs from each Barts Hospital (London, UK) and Tianjin University Hospital (Tianjin, China) were used to train and evaluate the supervised deep learning module. For training Fully Convolutional Networks (FCNs), WSIs manually annotated for both GCs and sinuses formed a ground-truth set and three FCNs were implemented: (i) a standard U-Net architecture; (ii) a U-Net model with an attention gate mechanism; and (iii) a multiscale-U-Net network (MS U-Net) that encodes, in parallel, a feature representation of the image at multiple resolutions. The MS U-Net achieved the best performance with an average dice score of 0.86 for GCs and 0.74 for sinuses. In comparison, the average dice score amongst four pathologists assessing 24 LN WSI for GCs and sinuses was 0.67 and 0.61, respectively, demonstrating the robustness of the smuLymphNet framework. To establish associations between morphometric immune features and patients’ outcomes, we assessed smuLymphNet captured GCs and sinuses from 686 WSIs from 96 TNBC patients with extensive longitudinal outcome data. We found significant morphological differences in involved and cancer-free LNs between N0 and N+ patients, with the latter displaying larger GCs with more irregular shapes, especially in their involved LNs. Moreover, in alignment with our previously published studies, our multiscale smuLymphNet framework recapitulated and extended the prognostic value of the assessment of GC formation in TNBC N0 patients. We further revealed, for the first time, the prognostic significance of the intranodal lymphatic sinuses when measured in their totality in involved LNs, and the association of alterations in subcapsular sinus areas with superior distant metastasis-free survival in cancer-free and involved LNs in TNBC N+ patients. In summary, smuLymphNet presents a robust multiscale deep learning framework to automatically detect, localise and quantify histopathological immune features in WSI of LNs. By applying smuLymphNet to LNs of TNBC patients from clinical trials, and thereby further evaluating its clinical utility, smuLymphNet could be implemented into the diagnostic digital pathology workflow and, as such, aid in informing on a patient’s disease trajectory. Citation Format: Gregory Verghese, Mengyuan Li, Fangfang Liu, Amit Lohan, Nikhil Cherian, Patrycja Gazinska, Aekta Shah, Aasiyah Oozeer, Cheryl Gillett, Elena Alberts, Thomas Hardiman, Roberto Salgado, Samantha Jones, Louise Jones, Selvam Thavaraj, Sarah E. Pinder, Swapni Rane, Amit Sethi, Anita Grigoriadis. Multiscale Deep Learning framework to capture systemic immune features in lymph nodes predictive of triple negative breast cancer outcome [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-01-01.

Abstract 6233: A deep learning pipeline to capture the prognostic immune responses in lymph nodes of breast cancer patients

Abstract Capturing tumor infiltrating leucocytes (TILS) and systemic immune responses in breast cancer informs disease progression and optimal treatment management. We have previously shown that morphological alterations in axillary lymph nodes (LNs), namely the formation of germinal centers in cancer-free LNs, adds prognostic value to TILs in triple negative breast cancer patients (TNBC) for the development of distant metastasis. Extending manual assessment of LNs beyond the detection of cancer requires the integration of robust deep learning pipelines into the digital pathology workflow. In this retrospective study, we used 1,100 Haematoxylin &amp; Eosin-stained (H&amp;E) Whole Slide Images (WSI) from Guy’s Hospital (London, UK) of metastatic and cancer-free LNs from 151 patients (100 N+) enriched for triple-negative or HER2-positive breast cancer to implement a supervised deep learning pipeline. A subset of 114 WSI, along with 5 breast cancer LN WSIs from each of Barts Hospital (London, UK) and Tianjin University Hospital (Tianjin, China), and 5 head and neck squamous cell carcinomas LN WSI (Guy’s Hospital) were used to develop, train and evaluate the segmentation task. For training Fully Convolutional Networks (FCNs), WSIs manually annotated for both germinal centers and sinuses formed a ground-truth set. Three FCNs were implemented: (i) a standard U-Net architecture; (ii) a U-Net model with an attention gate mechanism; and (iii) a multiscale-U-Net network (MSA-U-Net) that encodes, in parallel, a feature representation of the image at multiple resolutions. The MSA-U-Net achieved the best performance with an average dice score of 0.85 for germinal centers and 0.75 for sinuses. In comparison, the average dice score amongst 4 pathologists assessing 25 LN WSI for germinal centers and sinuses, was 0.67 and 0.61 respectively, demonstrating the robustness of the MSA-U-Net model. To quantify germinal centers and sinuses in LNs across the entire cohort, the trained MSA-U-Net was used in an inference step on all 1,100 WSI. The detected morphological features were initially localized within LNs using image thresholding and contouring techniques, and quantitatively assessed based on their number, area, shape, and Shannon diversity. We found significant morphological differences in metastatic and cancer-free LNs between N0 and N+ patients, with the latter displaying larger germinal centers with more irregular shapes especially in their metastatic LNs. In addition, we found differences in the Sinus area between LNs containing GCs and those without. Here, we propose a robust deep learning pipeline based on a multiscale FCN framework to automatically detect, localize and quantify histopathological immune features in WSI of LNs. By applying our pipeline to LNs of cancer patients, such as breast or head and neck, in prospective studies or clinical trials, we will further evaluate their prognostic and predictive values. Citation Format: Gregory Verghese, Mengyuan Li, Amit Lohan, Nikhil Cherian, Swapnil Rane, Fangfang Liu, Aekta Shah, Pat Gazinska, Selvam Thavaraj, Amit Sethi, Anita Grigoriadis. A deep learning pipeline to capture the prognostic immune responses in lymph nodes of breast cancer patients [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 6233.

Inhibitory effect of α-cubebenoate on atopic dermatitis-like symptoms by regulating Th2/Th1/Th17 balance in vivo

Ethnopharmacological relevanceBakumijiogan (Kampo herbal formulation) and Kangqian decoction (Chinese herbal medicine formulation) have been used for the treatment of atopic dermatitis (AD) like symptoms. Schisandra chinensis Baill (Family: Magnoliaceae) is a component of both formulations. Its extracts showed inhibition of AD. Aim of the studyWe aimed to elucidate an active phytochemical from Schisandra chinensis and evaluated its effects on AD-like symptoms. Materials and methodsWe fractionated a component from Schisandra chinensis by chasing inhibitory activity on mast cell degranulation. We identified α-cubebenoate as an active phytochemical and investigated its effects by using an in vivo 1-chloro-2,4-dinitrobenzene (CDNB)-induced AD model in BALB/c mice. Resultsα-Cubebenoate significantly decreased CDNB-induced skin hypertrophy and accumulation of mast cells in the epidermis and dermis. Increases in pro-inflammatory chemokine and cytokine levels in the skin, lymph node size, and immunoglobulin E levels in the serum were significantly ameliorated by α-cubebenoate. Conclusionα-Cubebenoate regulates dermal immune responses by suppressing the Th2/Th17/Th1 immune balances, resulting in amelioration of AD-like symptoms and suppression of immune response in lymph nodes. Thereby, this study provides evidence for its therapeutic efficacy in the treatment of AD symptoms.

Information flow in the spatiotemporal organization of immune responses.

Immune responses must be rapid, tightly orchestrated, and tailored to the encountered stimulus. Lymphatic vessels facilitate this process by continuously collecting immunological information (ie, antigens, immune cells, and soluble mediators) about the current state of peripheral tissues, and transporting these via the lymph across the lymphatic system. Lymph nodes (LNs), which are critical meeting points for innate and adaptive immune cells, are strategically located along the lymphatic network to intercept this information. Within LNs, immune cells are spatially organized, allowing them to efficiently respond to information delivered by the lymph, and to either promote immune homeostasis or mount protective immune responses. These responses involve the activation and functional cooperation of multiple distinct cell types and are tailored to the specific inflammatory conditions. The natural patterns of lymph flow can also generate spatial gradients of antigens and agonists within draining LNs, which can in turn further regulate innate cell function and localization, as well as the downstream generation of adaptive immunity. In this review, we explore how information transmitted by the lymph shapes the spatiotemporal organization of innate and adaptive immune responses in LNs, with particular focus on steady state and Type-I vs. Type-II inflammation.

Targeted delivery of regulatory macrophages to lymph nodes interferes with Tcell priming by preventing the formation of stable immune synapses.

Immunosuppressive myeloid cells are frequently induced in tumors and attenuate anti-tumor effector functions. In this study, we differentiate immunosuppressive regulatory macrophages (Mregs) from hematopoietic progenitors and test their potential to suppress adaptive immune responses in lymph nodes. Targeted delivery of Mregs to lymph nodes is facilitated by retroviral overexpression of the chemokine receptor CCR7 and intra-lymphatic cell application. Delivery of Mregs completely abolishes the priming of cognate CD8 cells and strongly reduces delayed-type hypersensitivity reactions. Mreg-mediated Tcell suppression requires cell-cell contact-regulated nitric oxide production. Two-photon microscopy reveals that nitric oxide produced by Mregs reduces the interaction duration between dendritic cells and Tcells. Exposure of activated Tcells to nitric oxide strongly reduces their binding to ICAM-1, indicating that nitrosylation of proteins involved in cell adhesion affects synapse formation. Thus, this study identifies a mechanism of myeloid cell-mediated immune suppression and provides an approach for its therapeutic use.

Detergent wash improves vaccinated lymph node handling ex vivo

Lymph nodes (LNs) are essential secondary immune organs where the adaptive immune response is generated against most infections and vaccines. We recently described the use of live ex vivo LN slices to study the dynamics of adaptive immunity. However, when working with reactive lymph nodes from vaccinated animals, the tissues frequently became dislodged from the supportive agarose matrix during slicing, leading to damage that prevented downstream analysis. Because reactive lymph nodes expand into the surrounding adipose tissue, we hypothesized that dislodging was a result of excess lipids on the collagen capsule of the LN, and that a brief wash with a mild detergent would improve LN interaction with the agarose without damaging tissue viability or function. Therefore, we tested the use of digitonin on improving slicing of vaccinated LNs. Prior to embedding, LNs were quickly dipped into a digitonin solution and washed in saline. Lipid droplets were visibly removed by this procedure. A digitonin wash step prior to slicing significantly reduced the loss of LN during slicing from 13 to 75% to 0–25%, without substantial impact on viability. Capture of fluorescent microparticles, uptake and processing of protein antigen, and cytokine secretion in response to a vaccine adjuvant, R848, were all unaffected by the detergent wash. This novel approach will enable ex vivo analysis of the generation of adaptive immune response in LNs in response to vaccinations and other immunotherapies.

Distributed Adaptive Search in T Cells: Lessons From Ants.

There are striking similarities between the strategies ant colonies use to forage for food and immune systems use to search for pathogens. Searchers (ants and cells) use the appropriate combination of random and directed motion, direct and indirect agent-agent interactions, and traversal of physical structures to solve search problems in a variety of environments. An effective immune response requires immune cells to search efficiently and effectively for diverse types of pathogens in different tissues and organs, just as different species of ants have evolved diverse search strategies to forage effectively for a variety of resources in a variety of habitats. Successful T cell search is required to initiate the adaptive immune response in lymph nodes and to eradicate pathogens at sites of infection in peripheral tissue. Ant search strategies suggest novel predictions about T cell search. In both systems, the distribution of targets in time and space determines the most effective search strategy. We hypothesize that the ability of searchers to sense and adapt to dynamic targets and environmental conditions enhances search effectiveness through adjustments to movement and communication patterns. We also suggest that random motion is a more important component of search strategies than is generally recognized. The behavior we observe in ants reveals general design principles and constraints that govern distributed adaptive search in a wide variety of complex systems, particularly the immune system.

Oral neonatal antibiotic treatment perturbs gut microbiota and aggravates central nervous system autoimmunity in Dark Agouti rats

Gut microbiota dysbiosis has been considered the essential element in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Antibiotics were administered orally to Dark Agouti (DA) rats early in their life with the aim of perturbing gut microbiota and investigating the effects of such intervention on the course of EAE. As a result, the diversity of the gut microbiota was reduced under the influence of antibiotics. Mainly, Firmicutes and Actinobacteria were replaced by Proteobacteria and Bacteroidetes, while decreased proportions of Clostridia and Bacilli classes were accompanied by an increase in Gamma-Proteobacteria in antibiotic-treated animals. Interestingly, a notable decrease in the Helicobacteraceae, Spirochaetaceae and Turicibacteriaceae was scored in antibiotic-treated groups. Also, levels of short chain fatty acids were reduced in the faeces of antibiotic-treated rats. Consequently, aggravation of EAE, paralleled with stronger immune response in lymph nodes draining the site of immunization, and increased inflammation within the CNS, were observed in antibiotic-treated DA rats. Thus, the alteration of gut microbiota leads to an escalation of CNS-directed autoimmunity in DA rats. The results of this study indicate that antibiotic use in early life may have subsequent unfavourable effects on the regulation of the immune system.

Impact of Precipitation of Antibody Therapeutics After Subcutaneous Injection on Pharmacokinetics and Immunogenicity

Antibody therapeutics with poor solubility in the subcutaneous matrix may carry unintended risks when administered to patients. The objective of this work was to estimate the risk of antibodies that precipitate in vitro at neutral pH by determining the impact of poor solubility on distribution of the drug from the injection site as well as immunogenicity in vivo. Using fluorescence imaging in a mouse model, we show that one such precipitation-prone antibody is retained at the injection site in the subcutaneous space longer than a control antibody. In addition, we demonstrate that retention at the injection site through aggregation is concentration-dependent and leads to macrophage association and germinal center localization. Although there was delayed disposition of the aggregated antibody to draining lymph nodes, no overall impact on the immune response in lymph nodes, systemic exposure of the antibody, or enhancement of the anti-drug antibody response was evident. Unexpectedly, retention of the precipitated antibody in the subcutaneous space delayed the onset of the immune response and led to an immune suppressive response. Thus, we conclude that precipitation due to poor solubility of high doses of antibody formulations delivered subcutaneously may not be of special concern in terms of exposure or immunogenicity.

A surface charge dependent enhanced Th1 antigen-specific immune response in lymph nodes by transfersome-based nanovaccine-loaded dissolving microneedle-assisted transdermal immunization.

The efficient delivery of vaccines to draining lymph nodes and the induction of robust local immune responses are crucial for immunotherapy. Transdermal administration has been evidenced to facilitate the delivery of ingredients to lymph nodes. In this study, transfersomes with opposite surface charges were applied for antigen encapsulation and these were integrated with dissolving microneedles to investigate their effects on immune responses via transdermal immunization. The microneedles were easily inserted into mouse skin and achieved the local release of nanovaccines into the dermis through dissolution. Although anionic nanovaccines promoted cellular uptake via DC2.4, cationic nanovaccines exhibited stronger escape capacities from endocytic compartments, facilitating antigen processing via an MHC-I presentation pathway, and formed larger accumulations in lymph nodes. Compared with their anionic counterparts, the cationic nanovaccines more efficiently activated DC maturation and induced Th1 immunity; this was suggested by the significantly increased IgG2a/IgG1 ratio and elevated cytokine secretion from Th1 cells, without an enhancement in the Th2 response. Such an enhanced Th1 antigen-specific immune response in lymph nodes via a transdermal vaccine delivery platform is beneficial for potential immunotherapy approaches.

Aminated nanomicelles as a designer vaccine adjuvant to trigger inflammasomes and multiple arms of the innate immune response in lymph nodes.

In this study, we suggest a designer vaccine adjuvant that can mimic the drainage of pathogens into lymph nodes and activate innate immune response in lymph nodes. By the amination of multivalent carboxyl groups in poly-(γ-glutamic acid) (γ-PGA) nanomicelles, the size was reduced for rapid entry into lymphatic vessels, and the immunologically inert nanomicelles were turned into potential activators of inflammasomes. Aminated γ-PGA nanomicelles (aPNMs) induced NLRP3 inflammasome activation and the subsequent release of proinflammatory IL-1β. The NLRP3-dependent inflammasome induction mechanism was confirmed through enzyme (cathepsin B and caspase-1) inhibitors and NLRP3 knockout mice model. After the aPNMs were combined with a clinically evaluated TLR3 agonist, polyinosinic–polycytidylic acid sodium salt (aPNM-IC), they triggered multiple arms of the innate immune response, including the secretion of pro-inflammatory cytokines by both inflammasomes and an inflammasome-independent pathway and the included type I interferons.

Towards a Multiscale Model of Acute HIV Infection

Human Immunodeficiency Virus (HIV) infection of humans represents a complex biological system and a great challenge to public health. Novel approaches for the analysis and prediction of the infection dynamics based on a multi-scale integration of virus ontogeny and immune reactions are needed to deal with the systems’ complexity. The aim of our study is: (1) to formulate a multi-scale mathematical model of HIV infection; (2) to implement the model computationally following a hybrid approach; and (3) to calibrate the model by estimating the parameter values enabling one to reproduce the “standard” observed dynamics of HIV infection in blood during the acute phase of primary infection. The modeling approach integrates the processes of infection spread and immune responses in Lymph Nodes (LN) to that observed in blood. The spatio-temporal population dynamics of T lymphocytes in LN in response to HIV infection is governed by equations linking an intracellular regulation of the lymphocyte fate by intercellular cytokine fields. We describe the balance of proliferation, differentiation and death at a single cell level as a consequence of gene activation via multiple signaling pathways activated by IL-2, IFNa and FasL. Distinct activation thresholds are used in the model to relate different modes of cellular responses to the hierarchy of the relative levels of the cytokines. We specify a reference set of model parameter values for the fundamental processes in lymph nodes that ensures a reasonable agreement with viral load and CD4+ T cell dynamics in blood.

Analysis of histological and immunological parameters of metastatic lymph nodes from colon cancer patients reveals that T-helper 1 type immune response is associated with improved overall survival.

Lymph node (LN) involvement in colonic carcinoma (CC) is a grave prognostic sign and mandates the addition of adjuvant treatment. However, in light of the histological variability and outcomes observed, we hypothesized that patients with LN metastases (LNM) comprise different subgroups.We retrospectively analyzed the histological sections of 82 patients with CC and LNM. We studied various histological parameters (such as tumor grade, desmoplasia, and preservation of LN architecture) as well as the prevalence of specific peritumoral immune cells (CD8, CD20, T-bet, and GATA-3). We correlated the histological and immunological data to patient outcome.Tumor grade was a significant prognostic factor even in patients with LNM. So was the number of LN involved (N1/N2 stage). From the morphological parameters tested (LN extracapsular invasion, desmoplasia in LN, LN architecture preservation, and mode of metastases distribution), none was found to be significantly associated with overall survival (OS). The mean OS of CD8 low patients was 66.6 ± 6.25 versus 71.4 ± 5.1 months for CD8 high patients (P = 0.79). However, T-helper (Th) 1 immune response skewing (measured by Th1/Th2 ratio >1) was significantly associated with improved OS. For patients with low ratio, the median OS was 35.5 ± 5 versus 83.5 months for patients with high Th1/Th2 ratio (P = 0.001).The histological presentation of LNM does not entail specific prognostic information. However, the finding of Th1 immune response in LN signifies a protective immune response. Future studies should be carried to verify this marker and develop a strategy that augments this immune response during subsequent adjuvant treatment.

Abstract IA28: Immunological insights from tracking dynamic cellular behavior in lymph nodes and tissues

Abstract A unique feature of the vertebrate immune system is its dependency on the migratory capacity of individual immune cells for proper development, differentiation and functional execution in vivo. High-resolution dynamic live-animal imaging has allowed scientists to decipher novel immunological insights, which is aided by increasing imaging sophistication, availability of in vivo experimental models, and high throughput multi-parameter data collection and analyses. Intravital imaging, combined with mathematical modeling and other immunological tools, has revealed an asymmetric role for MHC interactions in modulating CD4+ vs. CD8+ T lymphocyte surveillance strategies for cognate antigens in lymph nodes (LNs) [1]. Furthermore, naïve CD8+ T cells are preferentially attracted in a CCR5-dependent manner to LN niche sites where cognate antigen recognition occurs between DCs and CD4+ T cells via CCL3 / CCL4 gradient [2]. Additional data suggest that naïve CD8+ T cells regulate transient expression of preformed inflammatory chemokine receptors via integrin engagement early upon entry into inflamed LNs, suggesting a context-specific orchestration of immune responses in LNs. Once activated in the LNs, T cells travel to various anatomic compartments for delivery of their acquired effector function. Recent imaging experiments revealed that extravascular, CNS-resident CX3CR1+ APCs dynamically extend their cellular processes across intact vessel wall into vascular lumen. The frequency of projections increased in experimental autoimmune encephalomyelitis (EAE), whereas the number of projections remained stable compared to baseline days after other tissue injury such as CNS tumor infiltration and aseptic spinal cord trauma [3]. This unique behavior by CNS parenchymal CX3CR1+ cells may represent their unexplored functional role in antigen sampling and immune cell recruitment across the BBB. Lastly, we reported a novel role for cyclin-dependent kinase 5 (Cdk5), a proline-directed serine-threonine kinase, in T cell activation and persistence [4]. Cdk5 enzymatic activity has been found to be aberrantly up-regulated in a wide spectrum of tumors. Disruption of Cdk5 activity sensitizes tumor cells to elimination in a T cell-dependent manner, a process that is associated with an attenuated response in PD-L1 up-regulation upon interferon-γ exposure. Our additional emerging data further support a synergistic role of anti-Cdk5 agents in modulating tumor sensitivity to immunotherapeutic approaches. In conclusion, the tissue microenvironment plays a critical role in regulating host immune cell tumor crosstalk, and recent new advances in imaging modalities and in vivo experimental methodologies provided new insights that hold promises to propel the field of cancer immunotherapy forward.

Fine-Needle Aspiration Biopsy of the Lymph Node: A Novel Tool for the Monitoring of Immune Responses after Skin Antigen Delivery.

Assessment of immune responses in lymph nodes (LNs) is routine in animals, but rarely done in humans. We have applied minimally invasive ultrasound-guided fine-needle aspiration of the LN to a before-and-after study of the immune response to intradermally delivered Ag in healthy volunteers (n = 25). By comparison with PBMCs from the same individual, LN cells (LNCs) were characterized by reduced numbers of effector memory cells, especially CD8(+) TEMRA cells (3.37 ± 1.93 in LNCs versus 22.53 ± 7.65 in PBMCs; p = 0.01) and a marked increased in CD69 expression (27.67 ± 7.49 versus 3.49 ± 2.62%, LNCs and PBMCs, respectively; p < 0.0001). At baseline, there was a striking absence of IFN-γ ELISPOT responses to recall Ags (purified protein derivative, Tetanus toxoid, or flu/EBV/CMV viral mix) in LN, despite strong responses in the peripheral blood. However, 48 h after tuberculin purified protein derivative administration in the ipsilateral forearm resulting in a positive skin reaction, a clear increase in IFN-γ ELISPOT counts was seen in the draining LN but not in PBMCs. This response was lost by 5 d. These data suggest that the low levels of effector memory cells in the LN may explain the low background of baseline ELISPOT responses in LNs as compared with PBMCs, and the appearance of a response after 48 h is likely to represent migration of effector memory cells from the skin to the LN. Hence, it appears that the combination of intradermal Ag administration and draining LN sampling can be used as a sensitive method to probe the effector memory T cell repertoire in the skin.

Gene expression profiles of hair and wool sheep reveal importance of Th2 immune mechanisms for increased resistance to.

Management of gastrointestinal parasites is a critical issue for sheep producers worldwide. Increases in the prevalence of drug-resistant worms have complicated parasite control and increased economic losses. Therefore, other methods of parasite control need to be assessed, including the use of genetically resistant animals in breeding programs. Hair sheep breeds such as the St. Croix have greater parasite resistance than conventional wool breeds. However, the immune mechanisms that control parasite resistance in hair or wool breeds have not yet been fully determined, and information on cytokine expression profiles for both wool sheep selected for increased resistance and hair sheep is limited. Our objective was to investigate gene expression differences in 24 parasite-resistant hair and 24 susceptible wool sheep to identify immune effectors associated with resistance to . One-half of the lambs were infected and sacrificed at 3 or 27 d after infection. Remaining lambs were not infected. Breed differences in expression of genes associated with Th1 and Th2 immune responses in lymph nodes and abomasal tissue were determined. Th2-associated genes included IL-4, IL-13, IL-5, IgE, the α chain of the IL-4 receptor, and the α chain of the high-affinity IgE receptor (FcεRI). Th1-associated genes included interferon gamma (IFN-γ), the p35 subunit of IL-12 (IL-12 p35), and the β1 and β2 chains of the IL-12 receptor (IL-12 Rβ1 and IL-12 Rβ2, respectively). In both hair and wool sheep, infection with resulted in greater expression of IgE, IL-13, IL-5, and IL-12 p35 and somewhat reduced expression of IFNγ in lymph nodes. In abomasal tissue, parasite infection resulted in greater IgE, IL-13, FcεRI, and IL-12 p35 expression in infected lambs compared with control lambs. Between breeds, hair sheep had a stronger Th2 response after infection than wool sheep, with increased expression of IgE and IL-13 and decreased expression of IFNγ in lymph nodes and increased expression of IL-13 and decreased expression of IL-12 p35 in abomasal tissue. Expression of IL-4 in lymph nodes did not differ between hair and wool lambs, and IL-4, IL-5, IL-12 Rβ1, and IL-12 Rβ2 expression was too low to measure at the times sampled in abomasal tissue.

A Dendritic-Cell-Stromal Axis Maintains Immune Responses in Lymph Nodes

Within secondary lymphoid tissues, stromal reticular cells support lymphocyte function, and targeting reticular cells is a potential strategy for controlling pathogenic lymphocytes in disease. However, the mechanisms that regulate reticular cell function are not well understood. Here we found that during an immune response in lymph nodes, dendritic cells (DCs) maintain reticular cell survival in multiple compartments. DC-derived lymphotoxin beta receptor (LTβR) ligands were critical mediators, and LTβR signaling on reticular cells mediated cell survival by modulating podoplanin (PDPN). PDPN modulated integrin-mediated cell adhesion, which maintained cell survival. This DC-stromal axis maintained lymphocyte survival and the ongoing immune response. Our findings provide insight into the functions of DCs, LTβR, and PDPN and delineate a DC-stromal axis that can potentially be targeted in autoimmune or lymphoproliferative diseases.

Regulatory T Cells Suppress the Late Phase of the Immune Response in Lymph Nodes through P-Selectin Glycoprotein Ligand-1

Regulatory T cells (Tregs) maintain tolerance toward self-antigens and suppress autoimmune diseases, although the underlying molecular mechanisms are unclear. In this study, we show that mice deficient for P-selectin glycoprotein ligand-1 (PSGL-1) develop a more severe form of experimental autoimmune encephalomyelitis than wild type animals do, suggesting that PSGL-1 has a role in the negative regulation of autoimmunity. We found that Tregs lacking PSGL-1 were unable to suppress experimental autoimmune encephalomyelitis and failed to inhibit T cell proliferation in vivo in the lymph nodes. Using two-photon laser-scanning microscopy in the lymph node, we found that PSGL-1 expression on Tregs had no role in the suppression of early T cell priming after immunization with Ag. Instead, PSGL-1-deficient Tregs lost the ability to modulate T cell movement and failed to inhibit the T cell-dendritic cell contacts and T cell clustering essential for sustained T cell activation during the late phase of the immune response. Notably, PSGL-1 expression on myelin-specific effector T cells had no role in T cell locomotion in the lymph node. Our data show that PSGL-1 represents a previously unknown, phase-specific mechanism for Treg-mediated suppression of the persistence of immune responses and autoimmunity induction.