Journal of immunological methods最新文献

{"title":"Humoral and cellular response of two different vaccines against SARS-CoV-2 in a group of healthcare workers: An observational study","authors":"Nejla Stambouli ,&nbsp;Khadija Bahrini ,&nbsp;Chihebeddine Romdhani ,&nbsp;Aicha Rebai ,&nbsp;Sana Boughariou ,&nbsp;Mohamed Zakraoui ,&nbsp;Bilel Arfaoui ,&nbsp;Sameh Seyli ,&nbsp;Yasmine Boukhalfa ,&nbsp;Riadh Battikh ,&nbsp;Mohamed Ben Moussa ,&nbsp;Iheb Labbene ,&nbsp;Mustpha Ferjani ,&nbsp;Hedi Gharssallah","doi":"10.1016/j.jim.2024.113665","DOIUrl":"10.1016/j.jim.2024.113665","url":null,"abstract":"<div><p>On March 13, 2021, Tunisia started a widespread immunization program against SARS-CoV-2 utilizing different vaccinations that had been given emergency approval. Herein, we followed prospectively a cohort of participant who received COVID-19 vaccine (Pfizer BioNTech and Sputnik-Gameleya V). The goal of this follow-up was to define the humoral and cellular immunological profile after immunization by assessing neutralizing antibodies and IFN- γ release. 26 vaccinated health care workers by Pfizer BioNTech (<em>n</em>=12) and Sputnik-Gameleya V (<em>n</em>=14) were enrolled from June to December 2021 in Military hospital of Tunis. All consenting participants were sampled for peripheral blood after three weeks of vaccination. The humoral response was investigated by the titer of anti-SARS-CoV-2 immunoglobulin G (IgG) antibodies to S1 protein. The CD4 and CD8 T cell responses were evaluated by the QuantiFERON® SARS-CoV-2 (Qiagen® Basel, Switzerland).</p><p>Regardless the type of vaccine, the assessment of humoral and cellular response following vaccination showed a strong involvement of the later with expression of IFN-γ as compared to antibodies secretion. Moreover, we showed that people with past SARS-CoV-2 infection developed high levels of antibodies than those who are not previously infected. However, no significant difference was detected concerning interferon gamma (IFN-γ) expression by CD4 and CD8 T cells in health care worker (HCW) previously infection or not with COVID-19 infection. Analysis of immune response according to the type of vaccine, we found that Pfizer BioNTech induced high level of humoral response (91.66%) followed by Sputnik-Gameleya V (64.28%). However, adenovirus vaccine gave a better cellular response (57.14%) than mRNA vaccine (41.66%). Regarding the immune response following vaccine doses, we revealed a significant increase of neutralizing antibodies and IFN-γ release by T cells in patients fully vaccinated as compared to those who have received just one vaccine.</p><p>Collectively, our data revealed a similar immune response between Pfizer BioNTech and Sputnik-Gameleya V vaccine with a slight increase of humoral response by mRNA vaccine and cellular response by adenovirus vaccine. It's evident that past SARS-CoV-2 infection was a factor that contributed to the vaccination's increased immunogenicity. However, the administration of full doses of vaccines (Pfizer BioNTech or Sputnik-Gameleya V) induces better humoral and cellular responses detectable even more than three months following vaccination.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"528 ","pages":"Article 113665"},"PeriodicalIF":2.2,"publicationDate":"2024-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0022175924000504/pdfft?md5=21fd80d7d711ab78dc8734feec6bfe78&pid=1-s2.0-S0022175924000504-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140136906","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An optimized non-T cell transfection system based on HEK293FT cells for CD3ζ phosphorylation and ubiquitination","authors":"Jiaqi Zheng ,&nbsp;Yuchuan Zhang ,&nbsp;Yiting Cai ,&nbsp;Wei Han ,&nbsp;Wei Chen","doi":"10.1016/j.jim.2024.113664","DOIUrl":"10.1016/j.jim.2024.113664","url":null,"abstract":"<div><p>CD3ζ is part of the T cell receptor (TCR)/CD3 complex that plays a critical role in antigen recognition and subsequent T cell activation. Understanding the mechanisms that regulate CD3ζ can provide new insights into the T cell-mediated immune responses. However, it is challenging to deliver exogenous genes into T cells for functional and mechanistic analyses. To this end, we established a non-T cell transfection system based on HEK293FT cells to screen for candidate regulatory proteins. The transfection was optimized using relatively high confluent cultures and the transfection reagent PolyJet™. Pervanadate (PV) treatment sustained tyrosine phosphorylation of CD3ζ, and facilitated the subsequent activation-dependent ubiquitination by E3 ligase Cbl-b in the HEK293FT system. Lck and Zap70 kinases enhanced the levels of phosphorylated CD3ζ in the presence of PV. We compared the effects of E3 ligases and the corresponding adaptor proteins on activation-dependent ubiquitination of CD3ζ in the PV-stimulated cells, and found that Cbl-b was most effective. Taken together, we have demonstrated that a non-T cell transfection system based on PV-treated HEK293FT cells could effectively mimic CD3ζ phosphorylation and ubiquitination and is a promising model for studying the role of CD3ζ signaling in T cell activation.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"528 ","pages":"Article 113664"},"PeriodicalIF":2.2,"publicationDate":"2024-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140131719","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":"Selection of positive controls and their impact on anti-drug antibody assay performance","authors":"Joshua A. Weiner ,&nbsp;Harini Natarajan ,&nbsp;Calum J. McIntosh ,&nbsp;Eun Sung Yang ,&nbsp;Misook Choe ,&nbsp;Cassidy L. Papia ,&nbsp;Katherine S. Axelrod ,&nbsp;Gabriela Kovacikova ,&nbsp;Amarendra Pegu ,&nbsp;Margaret E. Ackerman","doi":"10.1016/j.jim.2024.113657","DOIUrl":"10.1016/j.jim.2024.113657","url":null,"abstract":"<div><p>Development of assays to reliably identify and characterize anti-drug antibodies (ADAs) depends on positive control anti-idiotype (anti-id) reagents, which are used to demonstrate that the standards recommended by regulatory authorities are met. This work employs a set of therapeutic antibodies under clinical development and their corresponding anti-ids to investigate how different positive control reagent properties impact ADA assay development. Positive controls exhibited different response profiles and apparent assay analytical sensitivity values depending on assay format. Neither anti-id affinity for drug, nor sensitivity in direct immunoassays related to sensitivity in ADA assays. Anti-ids were differentially able to detect damage to drug conjugates used in bridging assays and were differentially drug tolerant. These parameters also failed to relate to assay sensitivity, further complicating selection of anti-ids for use in ADA assay development based on functional characteristics. Given this variability among anti-ids, alternative controls that could be employed across multiple antibody drugs were investigated as a more uniform means to define ADA detection sensitivity across drug products and assay protocols, which could help better relate assay results to clinical risks of ADA responses. Overall, this study highlights the importance of positive control selection to reliable detection and clinical interpretation of the presence and magnitude of ADA responses.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"528 ","pages":"Article 113657"},"PeriodicalIF":2.2,"publicationDate":"2024-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140119769","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 flow cytometry-based assay to determine the ability of anti-Streptococcus pyogenes antibodies to mediate monocytic phagocytosis in human sera","authors":"Elena Boero ,&nbsp;Martina Carducci ,&nbsp;Alexander J. Keeley ,&nbsp;Berlanda Scorza Francesco ,&nbsp;Miren Iturriza-Gómara ,&nbsp;Danilo Gomes Moriel ,&nbsp;Rossi Omar","doi":"10.1016/j.jim.2024.113652","DOIUrl":"10.1016/j.jim.2024.113652","url":null,"abstract":"<div><p><em>Streptococcus pyogenes</em>, commonly referred to as Group A <em>Streptococcus</em> (Strep A), causes a spectrum of diseases, with the potential to progress into life-threatening illnesses and autoimmune complications. The escalating threat of antimicrobial resistance, stemming from the prevalent reliance on antibiotic therapies to manage Strep A infections, underscores the critical need for the development of disease control strategies centred around vaccination. Phagocytes play a critical role in controlling Strep A infections, and phagocytosis-replicating assays are essential for vaccine development. Traditionally, such assays have employed whole-blood killing or opsonophagocytic methods using HL-60 cells as neutrophil surrogates. However, assays mimicking Fcγ receptors- phagocytosis in clinical contexts are lacking. Therefore, here we introduce a flow cytometry-based method employing undifferentiated THP-1 cells as monocytic/macrophage model to swiftly evaluate the ability of human sera to induce phagocytosis of Strep A. We extensively characterize the assay's precision, linearity, and quantification limit, ensuring robustness. By testing human pooled serum, the assay proved to be suitable for the comparison of human sera's phagocytic capability against Strep A. This method offers a valuable complementary assay for clinical studies, addressing the gap in assessing FcγR-mediated phagocytosis. By facilitating efficient evaluation of Strep A -phagocyte interactions, it may contribute to elucidating the mechanisms required for the prevention of infections and inform the development of future vaccines and therapeutic advancements against Strep A infections.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"528 ","pages":"Article 113652"},"PeriodicalIF":2.2,"publicationDate":"2024-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0022175924000371/pdfft?md5=d41e0a9c1f2aff6a404855a17b04cf82&pid=1-s2.0-S0022175924000371-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140056155","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Performance of Softcup® menstrual cup and vulvovaginal swab samples for detection and quantification of genital cytokines","authors":"Nashlin Pillay ,&nbsp;Gugulethu Favourate Mzobe ,&nbsp;Marothi Letsoalo ,&nbsp;Asavela Olona Kama ,&nbsp;Andile Mtshali ,&nbsp;Stanley Nzuzo Magini ,&nbsp;Nikkishia Singh ,&nbsp;Vani Govender ,&nbsp;Natasha Samsunder ,&nbsp;Megeshinee Naidoo ,&nbsp;Dhayendre Moodley ,&nbsp;Cheryl Baxter ,&nbsp;Derseree Archary ,&nbsp;Sinaye Ngcapu","doi":"10.1016/j.jim.2024.113656","DOIUrl":"https://doi.org/10.1016/j.jim.2024.113656","url":null,"abstract":"<div><p>Cytokines are important mediators of immunity in the female genital tract, and their levels may be associated with various reproductive health outcomes. However, the measurement of cytokines and chemokines in vaginal fluid samples may be influenced by a variety of factors, each with the potential to affect the sensitivity and accuracy of the assay, including the interpretation and comparison of data. We measured and compared cytokine milieu in samples collected via Softcup® menstrual cup versus vulvovaginal swabs. One hundred and eighty vulvovaginal swabs from CAPRISA 088 and 42 Softcup supernatants from CAPRISA 016 cohorts of pregnant women were used to measure the concentrations of 28 cytokines through multiplexing. Cytokines measured in this study were detectable in each of the methods however, SoftCup supernatants showed consistently, higher detectability, expression ratios, and mean concentration of cytokines than vulvovaginal swabs. While mean concentrations differed, the majority of cytokines correlated between SoftCup supernatants and vulvovaginal swabs. Additionally, there were no significant differences in a number of participants between the two sampling methods for the classification of genital inflammation. Our findings suggest that SoftCup supernatants and vulvovaginal swab samples are suitable for the collection of genital specimens to study biological markers of genital inflammatory response. However, the Softcup menstrual cup performs better for the detection and quantification of soluble biomarkers that are found in low concentrations in cervicovaginal fluid.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"528 ","pages":"Article 113656"},"PeriodicalIF":2.2,"publicationDate":"2024-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140041855","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":"Development and basic performance verification of a rapid homogeneous bioassay for agonistic antibodies against the thyroid-stimulating hormone receptor","authors":"Motoki Hoshina ,&nbsp;Shiomi Ojima ,&nbsp;Atsushi Kawasaki ,&nbsp;Kosuke Doi ,&nbsp;Satoshi Ohta ,&nbsp;Asuka Inoue ,&nbsp;Hiroshi Murayama","doi":"10.1016/j.jim.2024.113655","DOIUrl":"10.1016/j.jim.2024.113655","url":null,"abstract":"<div><p>Graves' disease is a type of autoimmune hyperthyroidism caused by thyroid-stimulating antibodies (TSAb).<span>¹</span> The combination of a porcine thyroid cell bioassay and cyclic adenosine monophosphate (cAMP) immunoassay (TSAb-enzyme immunoassay; EIA) is a clinically approved TSAb measurement method. Due to the requirement of multiple procedures and a long assay time of 6 h in the TSAb-EIA, a simplified and rapid assay is desired. Herein, we developed a rapid homogeneous TSAb bioassay (rapid-TSAb assay) using the human embryonic kidney cell line (HEK293), engineered to express the human thyroid-stimulating hormone receptor (TSHR), along with a cAMP-dependent luminescence biosensor. The measurement consists of three steps: thawing frozen cells, blood sample addition, and luminescence detection. The procedures can be conducted within 1 h. The World Health Organization International Standard TSAb (NIBSC 08/204) stimulated the cells co-expressing TSHR and cAMP biosensor. The intra- and inter-assay coefficients of variance were &lt; 10%. Stimulation activity using wild-type TSHR and chimeric TSHR (Mc4) almost completely correlated with the tested Graves' disease and normal samples. In the rapid-TSAb assay, the evaluation of 39 samples, including TSHR antibody-positive sera, yielded a sensitivity of 100.0% and a specificity of 90.9%, compared to the TSAb-EIA control. The rapid-TSAb assay enables simple and rapid measurement of TSAb and is promising for improving the diagnosis of autoimmune thyroid diseases.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"528 ","pages":"Article 113655"},"PeriodicalIF":2.2,"publicationDate":"2024-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0022175924000401/pdfft?md5=a9a969d72e09ad18af00d70242612903&pid=1-s2.0-S0022175924000401-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140049672","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An immunochromatographic test for serological diagnosis of scrub typhus","authors":"Shuhao Yan ,&nbsp;Qingyu Lu ,&nbsp;Qingyuan Tao ,&nbsp;Yawei Lu ,&nbsp;Bao Gao ,&nbsp;Sibo Wang ,&nbsp;Xusheng Cai ,&nbsp;Lele Ai ,&nbsp;Xiaohui Xiong ,&nbsp;Min Cao ,&nbsp;Weilong Tan","doi":"10.1016/j.jim.2024.113653","DOIUrl":"10.1016/j.jim.2024.113653","url":null,"abstract":"<div><p>A fluorescent immunochromatographic test (FM-ICT) was developed for rapid detection of anti-<em>Orientia tsutsugamushi</em> antibodies in serum samples. The FM-ICT was constructed based on the dual-antigen sandwich method. Truncated 56 kDa outer membrane protein of <em>O. tsutsugamushi</em> strain SJ, was expressed in <em>E. coli</em> and mixed with those of Ptan and Gillam strains. A thin line of the protein mixture was precisely sprayed across a nitrocellulose membrane making this the “Test” line. Polyclonal antibodies (pAbs) to <em>O.tsutsugamushi</em> were sprayed in another line across the membrane making this the “Control” line. Fluorescent microspheres conjugated 56 kDa proteins reacting with sample serum will be captured on the “Test” line if the sample contains antibodies to <em>O.tsutsugamushi</em>. Several experimental parameters were optimized. After optimizing the reaction procedure, the results are visible, within 6 min, with the naked eye under ultraviolet light. The limit of detection (LOD) was determined to be 7.63 ng/mL with prepared polyclonal antibodies. No cross-reaction was observed with sera samples from other febrile diseases. In clinical evaluations, the strips showed 94.92% sensitivity (106/112) and 93.75% specificity (56/60). The FM-ICT we developed will provide a new tool for on-site diagnosis of scrub typhus<em>.</em></p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"528 ","pages":"Article 113653"},"PeriodicalIF":2.2,"publicationDate":"2024-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140021938","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":"High-resolution epitope mapping of commercial antibodies to ANCA antigens by yeast surface display","authors":"John S. Poulton ,&nbsp;Sajan Lamba ,&nbsp;Meghan Free ,&nbsp;Gang Xi ,&nbsp;Elizabeth McInnis ,&nbsp;Gabrielle Williams ,&nbsp;Stephan T. Kudlacek ,&nbsp;David Thieker ,&nbsp;Brian Kuhlman ,&nbsp;Ronald Falk","doi":"10.1016/j.jim.2024.113654","DOIUrl":"10.1016/j.jim.2024.113654","url":null,"abstract":"<div><p>Epitope mapping provides critical insight into antibody-antigen interactions. Epitope mapping of autoantibodies from patients with autoimmune diseases can help elucidate disease immunogenesis and guide the development of antigen-specific therapies. Similarly, epitope mapping of commercial antibodies targeting known autoantigens enables the use of those antibodies to test specific hypotheses. Anti-Neutrophil Cytoplasmic Autoantibody (ANCA) vasculitis results from the formation of autoantibodies to multiple autoantigens, including myeloperoxidase (MPO), proteinase-3 (PR3), plasminogen (PLG), and peroxidasin (PXDN). To perform high-resolution epitope mapping of commercial antibodies to these autoantigens, we developed a novel yeast surface display library based on a series of &gt;5000 overlapping peptides derived from their protein sequences. Using both FACS and magnetic bead isolation of reactive yeast, we screened 19 commercially available antibodies to the ANCA autoantigens. This approach to epitope mapping resulted in highly specific, fine epitope mapping, down to single amino acid resolution in many cases. Our study also identified cross-reactivity between some commercial antibodies to MPO and PXDN, which suggests that patients with apparent autoantibodies to both proteins may be the result of cross-reactivity. Together, our data validate yeast surface display using maximally overlapping peptides as an excellent approach to linear epitope mapping.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"528 ","pages":"Article 113654"},"PeriodicalIF":2.2,"publicationDate":"2024-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0022175924000395/pdfft?md5=f98aa6a7206d8538a429d399c6eed074&pid=1-s2.0-S0022175924000395-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140021939","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A comprehensive method for the phenotypical and functional characterization of recalled human memory B and T cells specific to vaccine antigens","authors":"Czdari Lee,&nbsp;Imtisal Imran,&nbsp;Sara Thomas,&nbsp;Mahyar Nouri-Shirazi","doi":"10.1016/j.jim.2024.113650","DOIUrl":"10.1016/j.jim.2024.113650","url":null,"abstract":"<div><p>Current methodologies for assessing vaccine effectiveness and longevity primarily center on measuring vaccine-induced neutralizing antibodies in serum or plasma. However, these methods overlook additional parameters such as the presence of memory B cells, even as antibody levels wane, and the pivotal role played by memory T cells in shaping antigen-specific memory B cell responses. Several studies have employed a combination of polyclonal activators, such as CpG and R848, along with various cytokines to provoke the recall of memory B cells from peripheral blood mononuclear cells (PBMCs) into antibody-secreting cells (ASCs). Other studies have examined the use of live attenuated viruses to stimulate antigen-specific memory T cells within PBMCs into effector T cells that produce Th1/Th2 cytokines. However, these studies have not fully elucidated the distinct effects of these polyclonal activators on individual subsets, nor have they evaluated whether the vaccine antigen alone is sufficient to trigger the recall of memory T cells.</p><p>Thus, in this study, we directly compared the capacity of two B cell polyclonal activators to induce the transition of existing vaccine-specific memory cells present in peripheral blood samples into ASCs. Simultaneously, we also assessed the transition of existing memory T cells into effector subsets in response to vaccine antigens.</p><p>Our findings demonstrate that both polyclonal activator combinations, CpG with IL-6 and IL-15, as well as R848 with IL-2, effectively induce the terminal differentiation of memory B cells into ASCs. Notably, CpG treatment preferentially expanded naïve and non-class-switched B cells, while R848 expanded class-switched memory cells, plasmablasts, and plasma cells. Consequently, R848 treatment led to a greater overall production of total and antigen-specific IgG immunoglobulins. Additionally, the exposure of isolated PBMCs to vaccine antigens alone proved sufficient for recalling the rare antigen-specific memory T cells into effector subsets, predominantly consisting of IFN-γ-producing CD4 T cells and TNF-β-producing CD8 T cells.</p><p>This study not only establishes a rationale for the selection of methods to expand and detect antigen-specific lymphocyte subsets but also presents a means to quantify vaccine effectiveness by correlating serum antibody levels with preexisting memory cells within peripheral blood samples.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"527 ","pages":"Article 113650"},"PeriodicalIF":2.2,"publicationDate":"2024-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140012733","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":"Methodological optimisation of thymocyte isolation and cryopreservation of human thymus samples","authors":"Ruth R. Hagen ,&nbsp;Calvin Xu ,&nbsp;Hui-Fern Koay ,&nbsp;Igor E. Konstantinov ,&nbsp;Stuart P. Berzins ,&nbsp;Katherine Kedzierska ,&nbsp;Carolien E. van de Sandt","doi":"10.1016/j.jim.2024.113651","DOIUrl":"10.1016/j.jim.2024.113651","url":null,"abstract":"<div><p>Premature lymphocytes develop into non-autoreactive, mature naïve CD4⁺ or CD8⁺ T cells in the thymus before entering the circulation. However, in-depth characterization of human thymocyte development remains challenging due to limited availability of human thymus samples and the fragile nature of thymocyte populations. Thymocytes often do not survive cryopreservation and thawing procedures, especially the fragile CD4⁺CD8⁺ double positive population. It is generally recommended to use fresh human thymus tissue on the day of excision to avoid any biases in thymocyte composition. This hampers the possibility to perform multiple experiments on the same thymus sample. To establish how the thymocyte viability and composition can be maintained, we compared two thymocyte isolation methods used for human and/or mice thymi, three cryopreservation methods in combination with our most gentle thawing technique. Based on our findings we established that fresh human thymi remain viable in cold storage for up to two days post-surgery without compromising thymocyte composition. Thymocytes can be cryopreserved if required, although the CD4⁺CD8⁺ double positive populations may be reduced. Our study provides thoroughly optimized methods to study human thymocyte development over a considerable time-frame post-surgery.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"528 ","pages":"Article 113651"},"PeriodicalIF":2.2,"publicationDate":"2024-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S002217592400036X/pdfft?md5=b7bd5746139388942901976709b6252d&pid=1-s2.0-S002217592400036X-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139990283","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}