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  • 1. Arestrom, Irene
    et al.
    Zuber, Bartek
    Bengtsson, Theresa
    Ahlborg, Niklas
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut, Avdelningen för immunologi.
    Measurement of human latent transforming growth factor beta 1 using a latency associated protein reactive elisa2012Ingår i: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 379, nr 1-2, s. 23-29Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Human Transforming Growth Factor (TGF)-beta 1, one of three TGF-beta isoforms, is a pleotropic cytokine critical for many physiological and immunological processes. TGF-beta 1 is secreted in a latent form, linked to Latency Associated Protein (LAP). Analysis of Latent TGF-beta 1 by TGF-ELISA requires dissociation of TGF-beta 1 from LAP, e.g. by acidification of samples. The ELISA then measures total TGF-beta 1, equivalent to dissociated Latent TGF-beta 1 plus any free TGF-beta 1 present prior to acidification. Evolutionary conservation of TGF-beta 1 across mammals also renders TGF-beta 1 ELISAs reactive with TGF-beta 1 in bovine serum often used in human cell cultures. To enable a direct analysis of Latent TGF-beta 1, monoclonal antibodies were made against LAP from human latent TGF-beta 1 and used to develop a LAP ELISA detecting Latent TGF-beta 1. The ELISA did not react with LAP from human Latent TGF-beta 2 or 3, respectively, nor with Latent TGF-beta in bovine serum. EDTA-containing plasma from healthy subjects (n = 20) was analyzed by conventional TGF-beta 1 ELISA and LAP ELISA. By TGF-beta 1 ELISA, total TGF-beta 1 were detected in all samples (median 133 pM, range 34-348 pM); low levels of free TGF-beta 1 found in 8/20 non-addified samples showed that >98.5% of the total TGF-beta 1 derived from Latent TGF-beta 1. Latent TGF-beta 1 found in non-acidified samples by LAP ELISA (median 154 pM, range 48-403 pM) was comparable in molar levels to, and correlated with, total TGF-beta 1 (r(s) 0.96, p<0.0001). A similar agreement between the total TGF-beta 1 and the LAP ELISA was found with citrate- and heparin-containing plasma. The LAP ELISA facilitates analysis of Latent TGF-beta 1 without sample acidification and is not compromised by the presence of bovine serum in human cell supernatants.

  • 2. Holthaus, Lisa
    et al.
    Lamp, Daniel
    Gavrisan, Anita
    Sharma, Virag
    Ziegler, Anette-Gabriele
    Jastroch, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. German Research Center for Environmental Health, Germany.
    Bonifacio, Ezio
    CD4(+) T cell activation, function, and metabolism are inhibited by low concentrations of DMSO2018Ingår i: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 463, s. 54-60Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Dimethyl sulfoxide (DMSO) is a polar organic solvent used in a wide range of biological applications. DMSO is routinely used as a cryoprotectant for long-term cell freezing as well as to dissolve peptides and drugs for immune cell functional assays. Here, human CD4(+) T cell activation, cytokine production, proliferation, and metabolism were investigated after stimulation in the presence of 0.01% to 1%, DMSO, representing concentrations commonly used in vitro. Surface expression of the activation markers CD69, CD25 and CD154 after polyclonal activation of CD4(+) T cells was inhibited by 0.25% or higher concentrations of DMSO. The frequencies of IL-21(+), IL-4(+), and IL-22(+) CD4(+) T cells, following polyclonal activation were variably inhibited by DMSO at concentrations ranging from 0.25% to 1%, whereas IFN gamma(+) cells were unaffected. CD4(+) T cell proliferation after anti-CD3 or antigen stimulation was inhibited by 0.5% DMSO and abolished by 1% DMSO. After polyclonal stimulation, glucose uptake was inhibited in the presence of 1% DMSO, but only minor effects on CD4+ T cell respiration were observed. Consistent with the immune effects, the gene expression of early signaling and activation pathways were inhibited in CD4+ T cells in the presence of 1% DMSO. Our study revealed that DMSO at concentrations generally used for in vitro studies of T cells impacts multiple features of T cell function. Therefore, we urge care when adding DMSO-containing preparations to T cell cultures.

  • 3. Huang, Jenny
    et al.
    Ehrnfelt, Cecilia
    Paulie, Staffan
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut, Avdelningen för immunologi. Mabtech AB, Sweden.
    Zuber, Bartek
    Ahlborg, Niklas
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut, Avdelningen för immunologi. Mabtech AB, Sweden.
    ELISpot and ELISA analyses of human IL-21-secreting cells: Impact of blocking IL-21 interaction with cellular receptors2015Ingår i: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 417, s. 60-66Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Interleukin (IL)-21 is crucial for the regulation of lymphocytes and is implicated in autoimmune and other diseases. The relevance of being able to measure human IL-21 prompted us to develop ELISA and ELISpot assays for analysis of IL-21 levels and IL-21-producing cells, respectively. Monoclonal antibodies (mAbs) to IL-21 were made and ELISA and ELISpot assays were developed. The selected detection mAb also neutralized IL-21-mediated activation of human cells. Peripheral blood mononuclear cells (PBMCs) from healthy donors (n = 24) were stimulated polyclonally (phytohemagglutinin; PHA) or with antigen (Candida albicans extract and tetanus toxoid). Using ELISpot, high numbers of IL-21-producing cells were detected after PHA activation; lower but positive responses to antigen were seen in approximately 50% of the donors. In contrast, the ELISA detected IL-21 in supernatants from PHA-activated cells but not from antigen-stimulated cells. When analyzing IL-17A in parallel, PHA and antigens induced detectable responses in ELISpot as well as in ELISA. Hypothesizing that the lack of detectable IL-21 levels after antigenic stimulation was due to a combination of low frequencies of IL-21-secreting cells and consumption of IL-21 by cellular receptors during cell culture, PBMCs (n = 18) were stimulated in the presence of the neutralizing detection mAb. When preventing IL-21 from interacting with its receptor, increased IL-21 levels were found by ELISA after PHA activation and IL-21 could also be measured after antigen stimulation. ELISpot results were unaffected by the addition of the neutralizing mAb. In conclusion, IL-21 secreted by low frequencies of antigen-specific ex vivo-stimulated PBMC can be difficult to detect by ELISA but prevention of IL-21 interaction with its receptor leads to detectable IL-21 levels. In ELISpot, where the cytokine is captured by mAbs on a solid phase immediately upon secretion, blocking the receptor interaction does not affect the detection of IL-21-secreting cells.

  • 4. Höglind, Ankie
    et al.
    Areström, Irene
    Ehrnfelt, Cecilia
    Masjedi, Khosro
    Zuber, Bartels
    Giavedoni, Luis
    Ahlborg, Niklas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. Mabtech, Sweden.
    Systematic evaluation of monoclonal antibodies and immunoassays for the detection of Interferon-gamma and Interleukin-2 in old and new world non-human primates2017Ingår i: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 441, s. 39-48Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Non-human primates (NHP) provide important animal models for studies on immune responses to infections and vaccines. When assessing cellular immunity in NHP, cytokines are almost exclusively analyzed utilizing cross-reactive anti-human antibodies. The functionality of antibodies has to be empirically established for each assay/application as well as NHP species.

    A rational approach was employed to identify monoclonal antibodies (mAb) cross-reactive with many NHP species. Panels of new and established mAbs against human Interferon (IFN)-gamma and Interleukin (IL)-2 were assessed for reactivity with eukaryotically expressed recombinant IFN-gamma and IL-2, respectively, from Old (rhesus, cynomolgus and pigtail macaques, African green monkey, sooty mangabey and baboon) and New World NHP (Ma's night monkey, squirrel monkey and common marmoset).

    Pan-reactive mAbs, recognizing cytokines from all NHP species, were further analyzed in capture assays and flow cytometry with NHP peripheral blood mononuclear cells (PBMC). Pan-reactive mAb pairs for IFN-gamma well as IL-2 were identified and used in ELISA to measure IFN-gamma and IL-2, respectively, in Old and New World NHP PBMC supernatants. The same mAb pairs displayed high functionality in ELISpot and FluoroSpot for the measurement of antigen-specific IFN-gamma and IL-2 responses using cynomolgus PBMC Functionality of pan-reactive mAbs in flow cytometry was also verified with cynomolgus PBMC.

    The development of well-defined immunoassays functional with a panel of NHP species facilitates improved analyses of cellular immunity and enables inclusion in multiplex cytokine assays intended for a variety of NHP.

  • 5. Jahnmatz, Peter
    et al.
    Bengtsson, Theresa
    Zuber, Bartek
    Farnert, Anna
    Ahlborg, Niklas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. Mabtech AB, Sweden.
    An antigen-specific, four-color, B-cell FluoroSpot assay utilizing tagged antigens for detection2016Ingår i: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 433, s. 23-30Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The FluoroSpot assay, a variant of ELISpot utilizing fluorescent detection, has so far been used primarily for assessment of T cells, where simultaneous detection of several cytokines has allowed a more qualitative analysis of functionally distinct T cells. The potential to measure multiple analytes also presents several advantages when analyzing B cells. Our aim was to develop a B-cell FluoroSpot assay adaptable to studies of a variety of antigens. The assay utilizes anti-IgG antibodies immobilized in 96-well filter membrane plates. During cell culture, IgG antibodies secreted by antibody-secreting cells (ASCs) are captured in the vicinity of each of these cells and the specificity of single ASCs is defined using antigens for detection. The antigens were labeled with biotin or peptide tags enabling secondary detection with fluorophore-conjugated streptavidin or tag-specific antibodies. The assay, utilizing up to four different tag systems and fluorophores simultaneously, was evaluated using hybridomas and immunized splenocytes as ASCs. Assay variants were developed that could: i) identify multiple ASCs with different antigen specificities; ii) detect ASCs showing cross-reactivity with different but related antigens; and iii) define the antigen-specificity and, by including anti-IgG subclass detection reagents, simultaneously determine the IgG subclass of antibodies secreted by ASCs. As demonstrated here, the B-cell FluoroSpot assay using tag-based detection systems provides a versatile and powerful tool to investigate antibody responses by individual cells that can be readily adapted to studies of a variety of antigen-specific ASCs.

  • 6. Rasul, Abu E.
    et al.
    Nagy, Noemi
    Sohlberg, Ebba
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut, Avdelningen för immunologi.
    Adori, Monika
    Claesson, Hans-Erik
    Klein, George
    Klein, Eva
    Simultaneous detection of the two main proliferation driving EBV encoded proteins, EBNA-2 and LMP-1 in single B cells2012Ingår i: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 385, nr 1-2, s. 60-70Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Epstein Barr virus (EBV) is carried by almost all adults, mostly without clinical manifestations. Latent virus infection of B lymphocytes induces activation and proliferation that can be demonstrated in vitro. In healthy individuals, generation of EBV induced malignant proliferation is avoided by continuous immunological surveillance. The proliferation inducing set of the virally encoded genes is expressed exclusively in B cells in a defined differentiation window. It comprises nine EBV encoded nuclear proteins, EBNA 1-6, and three cell membrane associated proteins, LMP-1,2A and 2B, designated as latency Type III. Outside this window the expression of the viral genes is limited. Healthy carriers harbor a low number of B lymphocytes in which the viral genome is either silent or expresses one virally encoded protein, EBNA-1, latency Type I. In addition, EBV genome carrying B cells can lack either EBNA-2 or LMP-1, latency Type IIa or Type IIb respectively. These cells have no inherent proliferation capacity. Detection of both EBNA-2 and LMP-1 can identify B cells with growth potential. We devised therefore a method for their simultaneous detection in cytospin deposited cell populations. Simultaneous detection of EBNA-2 and LMP-1 was reported earlier in tissues derived from infectious mononucleosis (IM), postransplantation lymphoproliferative disorders (PTLD) and from humanized mice infected with EBV. We show for the first time the occurrence of Type IIa and Type IIb cells in cord blood lymphocyte populations infected with EBV in vitro. Further, we confirm the variation of EBNA-2 and LMP-1 expression in several Type III lines and that they vary independently in individual cells. We visualize that in Type III LCL, induced for plasmacytoid differentiation by IL-21 treatment, EBV protein expression changes to Type ha (EBNA-2 negative LMP-1 positive). We also show that when the proliferation of EBV infected cord blood lymphocyte culture is inhibited by the immunomodulator, PSK the majority of the cells express latency Type IIa pattern. These results show that by modifying the differentiation state, the proliferating EBV positive B cells can be curbed. Type IIa expression is a characteristic for EBV positive Reed-Sternberg (R/S) cells in EBV positive Hodgkin's lymphomas. For survival and proliferation, the R/S cells require the contribution of the in vivo microenvironment Consequently, Type IIa lines could not be established from Hodgkin's lymphoma in vitro. We propose that these experimental cultures can be exploited for study of the Type IIa cells.

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