Host immune analyses require specific reagents to identify cellular and soluble components of the immune system. These immune reagents are often species-specific. For horses, various immunological tools have been developed and tested by different initiatives during the past decades. This article summarizes the development of well characterized monoclonal antibodies (mAbs) for equine immune cells, immunoglobulin isotypes, cytokines, and chemokines.

There are extensive immunological reagents available for laboratory rodents and humans. However, for veterinary species there is a need for expansion of immunological toolkits, with this especially evident for marine mammals, such as cetaceans. In addition to their use in a research setting, immune assays could be employed to monitor the health status of cetaceans and serve as an adjunct to available diagnostic tests. Such development of specific and sensitive immune assays will enhance the proper care and stewardship of wild and managed cetacean populations. Our goal is to provide immune reagents and immune assays for the research community, clinicians, and others involved in care of bottlenose dolphins. This review will provide an update on our development of a bottlenose dolphin immunological toolkit. The future availability and continued development of these reagents is critical for improving wild and managed bottlenose dolphin population health through enhanced assessment of their responses to alterations in the marine environment, including pathogens, and improve our ability to monitor their status following vaccination.

Immune-related adverse events (irAEs) are major barriers of clinical management and further development of immune checkpoint inhibitors (ICIs) for cancer therapy. Therefore, biomarkers associated with the onset of severe irAEs are needed. In this study, we aimed to identify immune features detectable in peripheral blood and associated with the development of severe irAEs that required clinical intervention.
We used a 43-marker mass cytometry panel to characterize peripheral blood mononuclear cells from 28 unique patients with melanoma across 29 lines of ICI therapy before treatment (baseline), before the onset of irAEs (pre-irAE) and at the peak of irAEs (irAE-max). In the 29 lines of ICI therapy, 18 resulted in severe irAEs and 11 did not.
Unsupervised and gated population analysis showed that patients with severe irAEs had a higher frequency of CD4+ naïve T cells and lower frequency of CD16+ natural killer (NK) cells at all time points. Gated population analysis additionally showed that patients with severe irAEs had fewer T cell immunoreceptor with Ig and ITIM domain (TIGIT+) regulatory T cells at baseline and more activated CD38+ CD4+ central memory T cells (TCM) and CD39+ and Human Leukocyte Antigen-DR Isotype (HLA-DR)+ CD8+ TCM at peak of irAEs. The differentiating immune features at baseline were predominantly seen in patients with gastrointestinal and cutaneous irAEs and type 1 diabetes. Higher frequencies of CD4+ naïve T cells and lower frequencies of CD16+ NK cells were also associated with clinical benefit to ICI therapy.
This study demonstrates that high-dimensional immune profiling can reveal novel blood-based immune signatures associated with risk and mechanism of severe irAEs. Development of severe irAEs in melanoma could be the result of reduced immune inhibitory capacity pre-ICI treatment, resulting in more activated TCM cells after treatment.

Crassostrea virginica is a well-established bivalve species for biomonitoring persistent organic pollutants such as polycyclic aromatic hydrocarbons (PAH) in aquatic environments. Differing biomonitoring methods employing either wild oysters inhabiting sites of interest or naïve cultured oysters deployed to sites for extended periods can be used for site evaluations. However, important differences in total contaminant concentrations accumulated have been observed between the wild and transplanted groups. Furthermore, although rearing cultured triploid oysters is widely popular in commercial farming, the difference in contaminant bioaccumulation potential between triploid and diploid cultured oysters is vastly understudied, particularly for organic contaminants such as PAH. This study explores differences in PAH kinetics between transplanted triploid and diploid cultured oysters and wild oysters at a PAH-impacted site during a 6-week field exposure study using novel immunological techniques: antibody-based biosensor technology and immunofluorescence visualization. Conventional chemical analysis of oyster tissue was also conducted for comparison. While differences were observed in the oyster interstitial fluid between the wild and transplanted oysters throughout the study, whole tissue analysis revealed differing trends at each time point. Our findings suggest that insufficient equilibration time may contribute to the differences observed between groups. Furthermore, when combined with visual evidence via immunofluorescence, internal partitioning of contaminants may be an important determinant for total concentrations measured. A better understanding of the differences observed between wild and transplanted oyster groups is necessary for improved biomonitoring. Our study highlights the value in employing novel immunological techniques to explore possible mechanisms driving these differences.

Immunoaffinity chromatography (IAC) is a type of liquid chromatography that uses immobilized antibodies or related binding agents as selective stationary phases for sample separation or analysis. The strong binding and high selectivity of antibodies have made IAC a popular tool for the purification and analysis of many chemicals and biochemicals, including proteins. The basic principles of IAC are described as related to the use of this method for protein purification and analysis. The main factors to consider in this technique are also presented under a discussion of the general strategy to follow during the development of a new IAC method. Protocols, as illustrated using human serum albumin (HSA) as a model protein, are provided for the use of IAC in several formats. This includes both the use of IAC with traditional low-performance supports such as agarose for off-line immunoextraction and supports used in high-performance IAC for on-line immunoextraction. The use of IAC for protein analysis as a flow-based or chromatographic immunoassay is also discussed and described using HSA and a competitive binding assay format as an example. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Off-line immunoextraction by traditional immunoaffinity chromatography Basic Protocol 2: On-line immunoextraction by high-performance immunoaffinity chromatography Basic Protocol 3: Competitive binding chromatographic immunoassay.

Adoptive transfer of T cells is a burgeoning cancer therapeutic approach. However, the fate of the cells, once transferred, is most often unknown. We describe the first clinical experience with a non-invasive biomarker to assay the apoptotic cell fraction (ACF) after cell therapy infusion, tested in the setting of head and neck squamous cell carcinoma (HNSCC). A patient with HNSCC received autologous tumor-infiltrating lymphocytes (TILs) labeled with a perfluorocarbon (PFC) nanoemulsion cell tracer. Nanoemulsion, released from apoptotic cells, clears through the reticuloendothelial system, particularly the Kupffer cells of the liver, and fluorine-19 (19F) magnetic resonance spectroscopy (MRS) of the liver was used to non-invasively infer the ACF.
Autologous TILs were isolated from a patient in their late 50s with relapsed, refractory human papillomavirus-mediated squamous cell carcinoma of the right tonsil, metastatic to the lung. A lung metastasis was resected for T cell harvest and expansion using a rapid expansion protocol. The expanded TILs were intracellularly labeled with PFC nanoemulsion tracer by coincubation in the final 24 hours of culture, followed by a wash step. At 22 days after intravenous infusion of TILs, quantitative single-voxel liver 19F MRS was performed in vivo using a 3T MRI system. From these data, we model the apparent ACF of the initial cell inoculant.
We show that it is feasible to PFC-label ~70×1010 TILs (F-TILs) in a single batch in a clinical cell processing facility, while maintaining >90% cell viability and standard flow cytometry-based release criteria for phenotype and function. Based on quantitative in vivo 19F MRS measurements in the liver, we estimate that ~30% cell equivalents of adoptively transferred F-TILs have become apoptotic by 22 days post-transfer.
Survival of the primary cell therapy product is likely to vary per patient. A non-invasive assay of ACF over time could potentially provide insight into the mechanisms of response and non-response, informing future clinical studies. This information may be useful to developers of cytotherapies and clinicians as it opens an avenue to quantify cellular product survival and engraftment.

暂无摘要。

Here, we present a protocol combining co-immunoprecipitation (Co-IP) and immunofluorescence approaches with cell cycle stage synchronization to detect cell-cycle-specific complexes. We describe steps to synchronize cells at specific cell cycle stages using drugs. We then detail the preparation of cell extracts from synchronized cells and fractionation of the protein complexes with density centrifugation, followed by Co-IP with specific antibodies. Protein-protein interactions are confirmed by localization using immunofluorescence imaging. This protocol is helpful for visualizing the dynamics of protein complex assembly. For complete details on the use and execution of this protocol, please refer to Habu and Kim (2021).1.

Why the gut microbiome is critical for the success of checkpoint inhibitor cancer therapy is a question that remains unanswered, but progress has slowed. We argue that this lack of advancement is due to an unappreciated biological detail. Here, we show that the antibiotic cocktail used in seminal publications-all of which have used the C57BL/6 mouse strain-are bitter and not tolerated by other common mouse strains (ie, BALB/c and DBA/2). We write to alert readers of this important biological limitation that must be considered when planning cancer experiments investigating the gut microbiota, to prevent the unnecessary dehydration of experimental animals, and to save our colleagues valuable experimental time and resources.

MRI is a widely available clinical tool for cancer diagnosis and treatment monitoring. MRI provides excellent soft tissue imaging, using a wide range of contrast mechanisms, and can non-invasively detect tissue metabolites. These approaches can be used to distinguish cancer from normal tissues, to stratify tumor aggressiveness, and to identify changes within both the tumor and its microenvironment in response to therapy. In this review, the role of MRI in immunotherapy monitoring will be discussed and how it could be utilized in the future to address some of the unique clinical questions that arise from immunotherapy. For example, MRI could play a role in identifying pseudoprogression, mixed response, T cell infiltration, cell tracking, and some of the characteristic immune-related adverse events associated with these agents. The factors to be considered when developing MRI imaging biomarkers for immunotherapy will be reviewed. Finally, the advantages and limitations of each approach will be discussed, as well as the challenges for future clinical translation into routine clinical care. Given the increasing use of immunotherapy in a wide range of cancers and the ability of MRI to detect the microstructural and functional changes associated with successful response to immunotherapy, the technique has great potential for more widespread and routine use in the future for these applications.