Journal of immunological methods最新文献

{"title":"Immunogenicity of tislelizumab: clinical summary of ADA incidence and feasibility of assessing the suitability of validation cut points using statistical methods.","authors":"Boxiang Tao, Meng Wang, Yanfei Yang, Na Zhao, Meiling Tan, Xiaolei Xun, Chunxiang Zeng, Fan Wang","doi":"10.1016/j.jim.2025.113983","DOIUrl":"https://doi.org/10.1016/j.jim.2025.113983","url":null,"abstract":"<p><p>Anti-drug antibody (ADA) is a critical indicator of the immunogenicity of biotherapeutics. To investigate the immunogenicity of tislelizumab, a humanized anti-PD-1 (programmed cell death 1) antibody, 3815 patients were analyzed for ADA incidence. The incidence of ADA is reported for the first time as 19.2 %. For any immunogenicity study, appropriate cut points are essential to accurately distinguish true ADA-positive samples from varying backgrounds across populations. Apart from the standardized false positive rate (FPR) method, statistical methods may be more efficient and powerful in evaluating the appropriateness of cut points. However, a detailed protocol for this approach is not yet in consensus. Therefore, by utilizing extensive data from tislelizumab clinical trials, this study explored the feasibility of assessing the suitability of validation cut points (VCPs) using statistical methods. ADA response datasets from 21 clinical tumor populations were evaluated for distribution differences in comparison to the validation population using statistical methods. Fourteen datasets exhibited significant differences from the validation dataset in both medians and variances, with FPRs exceeding the 2-11 % range. Seven datasets only differed significantly in medians, with five having out-of-range FPRs and their positive rates narrowly affected by cut point alteration. The results showed that statistical methods can effectively assess the suitability of VCPs: in-study cut points are recommended for datasets with significantly different median and variance, while VCPs may be appropriate when only medians differ.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113983"},"PeriodicalIF":1.6,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145228251","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fluorescence immunochromatographic assay based on CdSe/ZnS quantum dots for the detection of OXA-48.","authors":"Mengying Lun, Yumei Chen, Hongliang Liu, Yanhua Qi, Aiping Wang, Jingming Zhou","doi":"10.1016/j.jim.2025.113984","DOIUrl":"10.1016/j.jim.2025.113984","url":null,"abstract":"<p><p>The production of carbapenemase is the primary mechanism of bacterial resistance to carbapenem antibiotics. This resistance seriously compromises the efficacy of carbapenem antibiotics in treating infections and poses a significant challenge to clinical anti-infective therapy. Oxacillinase-48 (OXA-48) is a class D carbapenemase, whose genes are usually located on transferable elements and can spread between different strains and genera. Therefore, from the perspective of infection control, effective monitoring and prevention of OXA-48 are imperative. In this study, CdSe/ZnS quantum dots (QDs) were coupled with an anti-OXA-48 monoclonal antibody (mAb) as a fluorescent signal probe to establish a quantum dot-based fluorescent immunochromatographic strip. The test strip contains a Test line (T-line) coated with an anti-OXA-48 monoclonal antibody and a Control line (C-line) coated with Staphylococcus protein A (SPA). Based on a double-antibody sandwich detection mode, it can complete highly sensitive detection of OXA-48 within 10 min. The visual detection limit of this test strip for OXA-48 recombinant protein was 3.13 ng/mL, and there was no cross-reaction with other common carbapenemases (IMP-1, NDM-1, KPC-2, VIM-2, OXA-23). It can be positioned as a rapid and reliable OXA-48 detection tool, providing a novel method for the rapid identification of OXA-48-producing resistant bacterial strains in clinical practice.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113984"},"PeriodicalIF":1.6,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145228254","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Measurement of cleaved high-molecular-weight kininogen in patients with hereditary angioedema due to C1-inhibitor deficiency: preanalytical and analytical optimization","authors":"Chiara Suffritti ,&nbsp;Roberta Gualtierotti ,&nbsp;Andrea Zanichelli ,&nbsp;Matteo Vidali ,&nbsp;Massimo Cugno","doi":"10.1016/j.jim.2025.113981","DOIUrl":"10.1016/j.jim.2025.113981","url":null,"abstract":"<div><div>High-molecular-weight kininogen (HK) is a plasma protein cleaved during the activation of contact system by the enzyme kallikrein, releasing the peptide bradykinin, potent angioedema mediator. Plasma measurement of cleaved HK allows evaluating <em>in vivo</em> contact system activation but, when its main natural inhibitor (C1-inhibitor) is deficient, conflicting results have been reported, possibly due to <em>in vitro</em> activation.</div><div>We collected blood samples from 18 patients with C1-inhibitor deficiency and 7 healthy controls using five different anticoagulant mixtures to find the one that could completely block <em>in vitro</em> activation of contact system, so that a snapshot of the <em>in vivo</em> scenario could be obtained. The ability to prevent contact system activation <em>in vitro</em> was evaluated in plasma samples measuring levels of cleaved HK by SDS-PAGE with precast gels and immunoblotting. Correlations between cleaved HK and functional C1-inhibitor were evaluated as well as assay reproducibility.</div><div>In healthy subjects, no differences in cleaved HK were found for the different anticoagulants. In patients with C1-inhibitor deficiency, we observed higher levels of cleaved HK in sodium citrate samples (median 60.5 %, IQR 51.9–61.6 %) than in samples collected in a mixture of protease inhibitors (median 49.2 %, IQR 48.1–52.0 %) (<em>P</em> = 0.001). Only in the latter, cleaved HK levels correlated with circulating functional C1-inhibitor levels (<em>P</em> = 0.005). Using precast gels, intra- and inter-assay coefficients of variation were &lt; 5 %.</div><div>In conclusion, for measuring cleaved HK in plasma from patients with C1-inhibitor deficiency, the use of anticoagulant with protease inhibitors and precast gels allows reliable evaluation of <em>in vivo</em> contact system activation.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"544 ","pages":"Article 113981"},"PeriodicalIF":1.6,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145212881","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A high-throughput multi-species platform using Biolayer Interferometry Immunosorbent Assay (BLI-ISA) as an alternative to indirect ELISA for vaccine development","authors":"Ti Lu,&nbsp;Md Shafiullah Parvej,&nbsp;Sean K. Whittier,&nbsp;Suhrid Maiti,&nbsp;Zackary K. Dietz,&nbsp;Debaki R. Howlader,&nbsp;Mst Nusrat Zahan,&nbsp;Satabdi Biswas,&nbsp;William D. Picking,&nbsp;Wendy L. Picking","doi":"10.1016/j.jim.2025.113980","DOIUrl":"10.1016/j.jim.2025.113980","url":null,"abstract":"<div><div>In vaccine development, the ELISA (Enzyme-Linked Immunosorbent Assay) is commonly used to compare the antibody titers of samples from several treatment groups. This often requires extensive sample preparation, manual labor, and long incubation and processing times. Biolayer Interferometry (BLI) has emerged as an alternative to the ELISA for the detection and quantification of antigen-specific antibodies in biological samples. However, the implementation of BLI as a replacement for the ELISA in vaccine development requires that experimental parameters are established for accurate and reproducible results. Here we give a general protocol for a biolayer interferometry immunosorbent assay (BLI-ISA) for the comparison of antigen-specific antibody levels in treatment group sera that uses secondary antibody binding responses as replacement for ELISA endpoint titers. BLI-ISA provides rapid relative measurements of antigen-specific antibody levels (in nm binding shift), yielding results consistent with ELISA endpoint titers (expressed in ELISA Units), thereby enabling reliable comparison across treatment groups within a fraction of the time required for conventional ELISA.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"544 ","pages":"Article 113980"},"PeriodicalIF":1.6,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145102864","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Peripheral blood and nasal swabs Type I and III IFNs signature in RSV positive infant with bronchiolitis.","authors":"Francesco Savino, Paola Montanari, Maddalena Dini, Anna Pau, Lorenzo Ferroglio, Sara Burdisso, Cristina Calvi, Anna Clemente, Stefano Gambarino, Ilaria Galliano, Massimiliano Bergallo","doi":"10.1016/j.jim.2025.113918","DOIUrl":"10.1016/j.jim.2025.113918","url":null,"abstract":"<p><p>Bronchiolitis is an acute lower respiratory tract infection mainly affecting children under 24 months, primarily caused by Respiratory Syncytial Virus (RSV). Interferons (IFNs), cytokines central to innate and adaptive immunity, are secreted in response to viral infections. In RSV bronchiolitis, Type I and III IFN Signatures modulate antiviral responses and impact disease severity. This study aimed to evaluate the correlation between Type I and III IFNs transcriptional signatures in blood and nasal swabs with clinical presentation. We analyzed transcription levels of Type I and III IFN Signatures using a PCR real-time TaqMan assay in blood and nasal swabs from children hospitalized for RSV bronchiolitis and from healthy controls (HC). Interferon score I was significantly higher in bronchiolitis patients at admission than at discharge and compared to HC, in both blood and nasal swabs (p < 0.0001). A strong positive correlation for IFN score I between the two sample types was found (p < 0.0001). Patients with severe disease (requiring intensive care) had lower IFN score I than those with milder disease, in both blood (p = 0.0005) and nasal swabs (p = 0.0078). IFN score III was down-regulated in bronchiolitis patients compared to HC in blood samples (p < 0.0001), while in nasal swabs, this down-regulation was evident only at discharge (p < 0.0001 vs admission; p = 0.0546 vs HC). This study enhances understanding of IFN signature dynamics in RSV bronchiolitis, emphasizing the importance of sample type and signature specificity, and suggests their potential use as prognostic tools to improve patient management.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113918"},"PeriodicalIF":1.6,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144784465","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Lactococcus lactis as a delivery system for surface displayed mimotopes of major peanut allergen Ara h 2.","authors":"Mojca Lunder, Jernej Luzar, Tim Ključevšek, Aleš Berlec, Borut Štrukelj, Maja Skerbinjek Kavalar, Ana Koren, Peter Korošec","doi":"10.1016/j.jim.2025.113921","DOIUrl":"10.1016/j.jim.2025.113921","url":null,"abstract":"<p><p>The prevalence of peanut allergy has been steadily increasing over the last decades. Among food allergies it shows the highest severity and is potentially fatal. Allergen Ara h 2 stands out as the main elicitor of allergic reactions. Research in the field of peanut immunotherapy still strives to improve the significant drawbacks of currently approved therapeutics. In this study we tested three epitope-like peptides as epitope-specific treatment. The epitope-like peptides (mimotopes) of major peanut allergen Ara h 2 were expressed as fusions with scaffold protein - lactococcal peptidoglycan binding domain of AcmA. Mimotope-decorated L. lactis and mimotope-AcmA fusion proteins were characterized. The mimotopes retained the ability to bind IgE from peanut-allergic patients' sera. They showed low basophil activation in peanut-allergic patients, except for mimotope DHPRYGP fused to AcmA suggesting its allergenic activity was partially preserved due to the highest similarity with Ara h 2 epitope. Mimotopes fused to AcmA can elicit T cell response; the increased secretion of IFN-γ suggests the shift of T cell immune response to non-allergic type 1. The use of recombinant L.lactis as a delivery system for surface displayed mimotopes represents a promising strategy that would enable delivery and expression of mimotopes on mucosal surfaces and at the same time beneficial immunomodulating properties.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113921"},"PeriodicalIF":1.6,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144799349","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CAR binding efficacy and off-target interactions for common CAR detection reagents","authors":"Ryan Sinapius,&nbsp;Jeremy Fisher,&nbsp;Christopher Manning,&nbsp;Amrik Singh","doi":"10.1016/j.jim.2025.113970","DOIUrl":"10.1016/j.jim.2025.113970","url":null,"abstract":"<div><h3>Background</h3><div>Chimeric Antigen Receptor (CAR)-T cell therapy is a highly innovative form of cell-based immunotherapy. To expand CAR-T therapies into additional disease indications, identification of novel tumor antigens and grafting of CARs on other types of immune cells, such as macrophages and natural killer (NK) cells are being pursued. Therefore, as this treatment modality continues to evolve, there is a need for highly specific detection reagents to interrogate CAR surface expression.</div></div><div><h3>Methods/Objectives</h3><div>This study uses flow cytometry to compare the ability of various commercially available CAR detection reagents' to detect CAR constructs containing distinct hinges, linker sequences, and scFv designs. Furthermore, interrogation off-target interactions were performed within a PBMC matrix.</div></div><div><h3>Results</h3><div>All antibody-based CAR detection reagents demonstrated specificity and robust signal in stable CAR-Jurkat cell lines and primary human CAR-T cells. Protein L only detected anti-CD20 (Leu16) scFv-based CAR; off-target non-CAR staining was observed in non-transduced human PBMCs. Detection using recombinant protein antigen provides clean results in single-stain samples for CD19 and BMCA proteins, though CD20 did not produce positive staining in anti-CD20 CARs. Additionally, in live human PBMCs, false-positive signals may be observed for antibody conjugates due to the binding of human Fc receptors to IgG.</div></div><div><h3>Conclusions</h3><div>CAR idiotype antibodies, recombinant antigens, and anti-linker antibodies were able to provide specific and robust detection of CAR-expressing cells. However, the utility of both idiotype antibodies and recombinant antigens is limited to CARs targeting specific antigens. The anti-linker antibodies enable universal and broad detection of CARs independent of target antigens.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"544 ","pages":"Article 113970"},"PeriodicalIF":1.6,"publicationDate":"2025-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144932472","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Immunogenicity, biodistribution, and toxicology evaluation of Mycobacterium tuberculosis ag85a plasmid DNA in cynomolgus monkeys, mice and guinea pigs","authors":"Danyang Zhang ,&nbsp;Yourong Yang ,&nbsp;Zhen Zhang ,&nbsp;Junxian Zhang ,&nbsp;Xueqiong Wu ,&nbsp;Yan Liang","doi":"10.1016/j.jim.2025.113969","DOIUrl":"10.1016/j.jim.2025.113969","url":null,"abstract":"<div><h3>Background</h3><div><em>Mycobacterium tuberculosis</em> (MTB) Ag85A has become a component of multiple new tuberculosis vaccines. It is necessary to evaluate the immunogenicity, biological distribution, and safety of <em>ag85a</em> plasmid DNA (pDNA) to lay the foundation for the design of new vaccines.</div></div><div><h3>Method</h3><div>Chronic toxicity test: cynomolgus monkeys were injected intramuscularly with different doses of <em>ag85a</em> pDNA, and the vaccine absorption kinetics, tissue distribution, and toxicity were observed. Their immune function was evaluated. Acute toxicity test: Mice were injected intramuscularly 0.5 ml saline, and injected intramuscularly and intravenously 0.5 mg/0.5 ml <em>ag85a</em> pDNAs, respectively. The toxicity and death of the mice were observed continuously for 14 days. Allergic test: Guinea pigs were intraperitoneally injected with different doses of <em>ag85a</em> pDNA. After stimulation, the allergic reaction and its severity were observed.</div></div><div><h3>Results</h3><div>Chronic and acute toxicity tests demonstrated that <em>ag85a</em> pDNA injections caused no clinical symptoms or tissue damage. Repeated intramuscular injections in cynomolgus monkeys enhanced specific Th1 immune responses, with pDNA rapidly entering the bloodstream and its concentration positively correlating with dosage. After 8 weeks, <em>ag85a</em> gene was detected only in muscles, myocardium, iliac lymph nodes, and blood. Guinea pig allergy tests showed no weight changes or allergic reactions, even after multiple sensitizations.</div></div><div><h3>Conclusions</h3><div>The <em>ag85a</em> pDNA showed good safety in cynomolgus monkeys, mice, and guinea pigs, and induced high levels of antibodies and T-cell responses, making it a candidate antigen for the construction of a new tuberculosis vaccine.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"544 ","pages":"Article 113969"},"PeriodicalIF":1.6,"publicationDate":"2025-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144957000","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In vitro and in vivo lipopolysaccharide-driven activation of human neutrophils in healthy volunteers as a tool for clinical drug development","authors":"Hendrika W. Grievink ,&nbsp;Coen Breedveld ,&nbsp;John Öhd ,&nbsp;Mark Schoonderwoerd ,&nbsp;Hjalmar P. Permentier ,&nbsp;Amanda C. Foks ,&nbsp;Ilze Bot ,&nbsp;Elsa Neubert ,&nbsp;Matthijs Moerland","doi":"10.1016/j.jim.2025.113968","DOIUrl":"10.1016/j.jim.2025.113968","url":null,"abstract":"<div><div>Neutrophils are an emerging target for therapeutical intervention in both autoimmune diseases as well as cancer. Since healthy humans lack constitutive neutrophil activation, induction of neutrophil activation is necessary to evaluate investigational compounds and can be achieved <em>via</em> intravenous administration of lipopolysaccharides (LPS). Furthermore, LPS stimulation can be performed <em>ex vivo</em> during clinical trials, and <em>in vitro</em> for pre-clinical analysis. Therefore, we aimed to provide a time course of neutrophil responses after <em>in vivo</em> LPS administration using samples from human endotoxemia trials and compared this to <em>in vitro</em> LPS stimulated whole blood cultures. We performed shotgun proteomics on <em>in vivo</em> stimulated neutrophils, and measured neutrophil activation by flow cytometry using CD11b and CD62L as activation markers and elastase, MPO, LL37 and nucleosome levels as degranulation and NETosis markers. Neutrophil numbers rapidly increased after LPS administration. In line, we found significant increases in neutrophil activation and degranulation markers both <em>in vitro</em> as well as <em>in vivo</em>, which all returned to baseline within 24 h. Degranulation proteins and NETosis related nucleosomes rapidly increased after LPS administration (1 h after exposure) <em>in vivo</em>, while higher concentrations of LPS were necessary <em>in vitro</em>. Lastly, shotgun proteomics revealed little but significant differences in the neutrophil proteome after <em>in vivo</em> LPS administration, pointing to degranulation after LPS stimulation. Both, the <em>in vitro</em> whole blood LPS stimulation assay and the human endotoxemia model, could be valuable tools for evaluation of the effects of future drugs modulating neutrophil responses during preclinical and clinical development.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"544 ","pages":"Article 113968"},"PeriodicalIF":1.6,"publicationDate":"2025-08-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144934090","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A novel variation of the mixed lymphocyte reaction for measuring T cell responses to skin-specific antigens of pigs","authors":"Elin Manell ,&nbsp;M. Esad Gunes ,&nbsp;Philip Jordache ,&nbsp;Satyajit Patwardhan ,&nbsp;Julie Hong ,&nbsp;David Sachs ,&nbsp;Joshua Weiner","doi":"10.1016/j.jim.2025.113920","DOIUrl":"10.1016/j.jim.2025.113920","url":null,"abstract":"<div><div>Skin and vascularized composite allografts (VCA) containing skin are transplanted to restore form and function of tissues after major injuries. Skin has long been recognized as being particularly immunogenic, causing high risk of rejection and immune sensitization. Due to skin-specific antigens, donor skin is often rejected even in animals that are tolerant of the remaining donor tissue. To study the reaction of lymphocyte subsets against these minor and/or tissue-specific skin antigens in swine made tolerant to allogeneic donors through hematopoietic stem cell transplants (HSCT), we developed a skin-adapted variation of the mixed lymphocyte reaction (MLR). We processed porcine skin into single cell suspensions to be used as stimulators. Peripheral blood mononuclear cells were used as responders. We first optimized the concentrations of skin stimulators to achieve T cell proliferation with minimal self-background reactivity. The assay was then tested in two pigs that had received combined VCA/HSCT. The first pig had not rejected any part of the VCA, and the second pig was actively rejecting the epidermis of the VCA at the time of the assay. Despite lack of anti-donor MLR responses against donor lymphocytes in the peripheral blood in either animal, the second pig demonstrated a specific response against donor skin cells. Our results suggest that the assay will be useful to study recipient sensitization against skin antigens, even in otherwise tolerant animals. This assay may have both diagnostic and therapeutic implications for immune responses specific to the skin.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"543 ","pages":"Article 113920"},"PeriodicalIF":1.6,"publicationDate":"2025-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144855492","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}