We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent ma... more We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent mammalian cells using fluorescence in situ hybridization (FISH) probes. The method, which we call FISH-Flow, allows for high-throughput multiparametric measurements of gene expression, a task that was not feasible with earlier, microscopy-based approaches. The FISH-Flow protocol involves cell fixation, permeabilization and hybridization with a set of fluorescently labeled oligonucleotide probes. In this protocol, surface and intracellular protein markers can also be stained with fluorescently labeled antibodies for simultaneous protein and mRNA measurement. Moreover, a semiautomated, single-tube version of the protocol can be performed with a commercially available cell-wash device that reduces cell loss, operator time and interoperator variability. It takes ~30 h to perform this protocol. An example of FISH-Flow measurements of cytokine mRNA induction by ex vivo stimulation of primed T cells with specific antigens is described.
Host responses to Mycobacterium tuberculosis infection provide a basis for diagnosis of latent in... more Host responses to Mycobacterium tuberculosis infection provide a basis for diagnosis of latent infection and active disease. T cell responses have been the mainstay for diagnosis of latent infection and may contribute to diagnosis of active disease. Recent advances in characterizing humoral responses will likely contribute to improved diagnosis of active disease. However, these measures fail to distinguish the continuum of infection states. Moving to a systems approach to biomarker discovery may provide the resolution that current methods of diagnosis lack. The chapter evaluates the current use of T cell and B cell responses for diagnosis and the limitations of applying them separately. The possibility that macrophage or monocyte activation may serve as a biomarker is also addressed. We consider whether methodologies that combine (a) multifunctional T cell responses and T cell types, (b) monocyte/macrophage characteristics that reveal response to infection, and (c) dominant B cell responses to M. tuberculosis growth-phase-specific antigens can further contribute to a systems approach for biomarker discovery that can distinguish among infection states.
Flow cytometric characterization of Ag-specific T cells typically relies on detection of protein ... more Flow cytometric characterization of Ag-specific T cells typically relies on detection of protein analytes. Shifting the analysis to detection of RNA would provide several significant advantages, which we illustrate by developing a new host immunity-based platform for detection of infections. Cytokine mRNAs synthesized in response to ex vivo stimulation with pathogen-specific Ags are detected in T cells with single-molecule fluorescence in situ hybridization followed by flow cytometry. Background from preexisting in vivo analytes is lower for RNAs than for proteins, allowing greater sensitivity for detection of low-frequency cells. Moreover, mRNA analysis reveals kinetic differences in cytokine expression that are not apparent at the protein level but provide novel insights into gene expression programs expected to define different T cell subsets. The utility of probing immunological memory of infections is demonstrated by detecting T cells that recognize mycobacterial and viral Ags in donors exposed to the respective pathogens.
Fluorescence in situ hybridization coupled with flow cytometry (FISH-Flow) is a highly quantitati... more Fluorescence in situ hybridization coupled with flow cytometry (FISH-Flow) is a highly quantitative, high-throughput platform allowing precise quantification of total mRNA transcripts in single cells. In undiagnosed infections posing a significant health burden worldwide, such as latent tuberculosis or asymptomatic recurrent malaria, an important challenge is to develop accurate diagnostic tools. Antigen-specific T cells create a persistent memory to pathogens, making them useful for diagnosis of infection. Stimulation of memory response initiates T-cell transitions between functional states. Numerous studies have shown that changes in protein levels lag real-time T-cell transitions. However, analysis at the single-cell transcriptional level can determine the differences. FISH-Flow is a powerful tool with which to study the functional states of T-cell subsets and to identify the gene expression profiles of antigen-specific T cells during disease progression. Advances in instrumentation, fluorophores, and FISH methodologies will broaden and deepen the use of FISH-Flow, changing the immunological field by allowing determination of functional immune signatures at the mRNA level and the development of new diagnostic tools.
We have identified and characterized the tissue distribution of the antigen recognized by a novel... more We have identified and characterized the tissue distribution of the antigen recognized by a novel monoclonal antibody (mAb) IBI0, raised against an activated yo T cell clone. Immunohistochemistry of tissue sections, and analysis of single cell suspensions by flow cytometry revealed that mAb IBI0 weakly reacted with <6% of normal human peripheral blood mononuclear cells (PBMC). After 5-6 days of in vitro culture of PBMC activated with phytohemagglutinin (PHA), 55% of the CD4+ and 25% of the CD8+ T cells became 1B1O+. IBI0 expression was maintained on long term cultured interleukin 2 (IL-2)-dependent T cell receptor (TCR) aW and yo+ clones, and importantly, in contrast to resting T cells, the majority of in vivo activated synovial T lymphocytes from a patient with rheumatoid arthritis were IBI0+. In addition, myelo-monocytic U927 cells, tissue macrophages and some epithelia and fibroblasts were found to react with mAb IBIO. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of molecules immuno-precipitated by mAb IBIO from radio-iodinated cell surface membrane lysates of T lymphocyte and U937 cells revealed 26 and 29 kiloDalton (kDa) glycoproteins respectively. In conclusion, mAb I BI a recognizes a novel «late» appearing 26 kDa T cell activation antigen that may be useful for further studies of activated T cells in health and disease. Abbreviations: FCS =fetal calf serum; FM =final medium; FIYC =fluoresceine isothiocyanate; LBC =lymphoblastoid B cells; IL =interleukin-2; IL-2R =IL-receptor; Ig =immunoglobulin; IV = international units; IMDM = Iscoves modified Dulbecco medium; kDa = kilodalton; MHC = major histo compatibility; mAb = monoclonal antibody; PBMC = peripheral blood mononuclear cells; pmst = phenyl methyl sulfonyl fluoride; PBS = phosphate buffered saline; R IL-2 = recombinant IL-2; SDS-PAGE = sodium dodecyl sulfate polyacrilamide gel electrophoresis; SRBC =sheep red blood cell; YCR =T cell receptor; YBS =tris buffered saline; RA = rheumatoid arthritis; VLA =very late antigen.°1 999 by URBAN & FISCHER
Establishing diagnosis of latent and active histoplasmosis is challenging. Interferon gamma-relea... more Establishing diagnosis of latent and active histoplasmosis is challenging. Interferon gamma-release assays (IGRAs) may provide evidence of latent and active infection.
Cross-linking of CD8 and HLA class I molecules with appropriate monoclonal antibodies (mAb) and g... more Cross-linking of CD8 and HLA class I molecules with appropriate monoclonal antibodies (mAb) and goat anti-mouse Ig (GaMlg) antibody resulted in a marked proliferation of resting human CD8 cells in the presence of interleukin-2 (IL-2). These cells also expressed IL-2 receptor (IL-2R), transferrin receptor, HLA-DR and-DQ antigens. Activation of the cross-linked CD8 cells is apparently independent of accessory monocytes. Various anti-CD8 and anti-HLA class I mAb recognizing nonpolymorphic antigenic determinants were examined for the efficacy of activating CD8 cells. Among mAb specific for HLA class I molecules, PA2.6, MB40.5, BB7.7, A 1.4, and W6/32 mAb markedly stimulated the proliferation of cross-linked CD8 cells, whereas BBM. 1, Ql/28, and HClO mAb were found inactive. Footprinting analysis of HLA class 1 molecules suggested that the activity of these anti-HLA class I mAb appeared to be related to the corresponding peptides they protect from enzymatic digestion. In contrast to the anti-HLA class I mAb, all anti-CD8 mAb examined (C8, OKT8A, and anti-Leu-2a) induced the proliferation of CDS-HLA class I cross-linked cells with similar efficacy. These results suggest that physical interaction between CD8 and at least one specific region of HLA class I molecules can trigger the activation of resting human CD8 cells.
Five monoclonal antibodies (A7, B24, I14, L12, and M2) recognizing different epitopes of the huma... more Five monoclonal antibodies (A7, B24, I14, L12, and M2) recognizing different epitopes of the human natural IFN-γ were prepared by immunizing BALB/c mice with a highly purified human natural IFN-γ preparation (107 U/mg). All five antibodies had high IFN-γ-binding activity but exhibited differential IFN-γ-neutralizing activities. Furthermore, none of them neutralized the antiviral activity exhibited by either IFN-α or IFN-β preparations, indicating thus their specificity for IFN-γ. The A7, L12, M2, and I14 monoclonal antibodies, but not the B24, blocked the augmentation of natural killer cytotoxicity, mediated by peripheral blood monocyte-depleted lymphocytes, by Escherichia coli-derived IFN-γ or natural IFN-γ but not by IFN-α2. All five monoclonal antibodies precipitated an identical molecular complex containing two major protein components with molecular weights of 20,000 (20 kD) and 25,000 (25 kD) and two minor components with molecular weights of 17,000 (17 kD) and 45,000 (45 kD). Treatment of the immun...
A murine monoclonal antibody (MAb) was obtained that showed unique specificity for the immunizing... more A murine monoclonal antibody (MAb) was obtained that showed unique specificity for the immunizing T-cell line HPB-ALL. This antibody, C37 (an IgG,,K) also reacted with a small (2-5%) population of normal peripheral blood T (PBL-T) cells. These C37-positive (C37+) cells were found in both the T4/Leu3+ and T8/Leu2+ subsets. Like OKT3 antibody, C37 induced T-cell mitogenesis with a peak proliferative response at day 3. In longterm cultures containing irradiated autologous feeder cells and IL-2, C37 antibody caused the selective expansion of C37+ T cells. On HPB-ALL cells C37 induced comodulation of the T3 molecule. C37 precipitated a disulfide-linked dimer characteristic of the T-cell antigen receptor consisting of an a-subunit (45-48 kD) and a ß-subunit (38-42 kD) from both C37+ T-cell blasts of a normal individual and HPB-ALL cells that were surface radioiodinated. However, the precipitated molecule isolated from C37 antibody-activated T-cell blasts exhibited a different pi from that isolated from HPB-ALL cells. Our studies indicate that C37 recognizes an epitope on the T-cell receptor molecule that is shared by a subpopulation of human T cells, which raises the possibility that multiple variable-region associated and/or framework-like determinants of the T-cell antigen receptor can be defined serologically and used in functional and molecular studies of T-cell subsets.
A T cell membrane antigen-recognition structure has recently been described ill several laborator... more A T cell membrane antigen-recognition structure has recently been described ill several laboratories, including our own (1-6). This molecule is a heterodimer of approximately 80-90 Kd, composed of two disulfide-linked chains. It fulfills many of the requirements for the T cell receptor for antigen, such as major histocompatibility complex-restricted antigen recognition (2-4). This molecule has been studied on murine cells (1, 2, 4), human T cell clones (3), and human T cell leukemia cells (5). The latter study, from this laboratory, described monoclonal antibodies reactive with idiotype-like determinants on a human T cell leukemia (5). Of two antibodies, one reacted with a private idiotype-like determinant and another cross-reacted with 1-2% of normal T cells by immunofluorescence. Both antibodies immunoprecipitated the same ~80 Kd disulfide-linked heterodimer from the membrane-iodinated leukemic cells, and this molecule comodulated with the T3 molecule. Thus, strong indirect evidence suggested that these monoclonal antibodies were recognizing a T cell antigen receptor. The presence of private idiotypic determinants and shared determinants on this molecule suggested a general structure similar to that of immunoglobulin molecules (7). This paper compares two T cell antiidiotypic antibodies each specifically reactive with their respective leukemia T cell clone. Immunoprecipitation with these antibodies revealed major differences between the two idiotype-bearing heterodimer molecules. In addition, proliferation studies in response to Sepharose-linked antiidiotypic antibody revealed marked differences between the two T cell leukemias. Materials and Methods Cell Preparations. Cells derived from the peripheral blood of a patient with Sezary syndrome, patient SU, and another patient with T celt chronic lymphocytic leukemia
We have previously described fluorescence in situ hybridization for mRNA detection combined with ... more We have previously described fluorescence in situ hybridization for mRNA detection combined with flow cytometry (FISH-Flow) as a novel method to detect gene expression at a single-cell level. Using mRNA as analyte in flow cytometry presents multiple, biological and technical advantages. Proof-of-principle experiments have also shown that FISH-Flow can detect rare events including antigen-specific T cell responses. Whether the method is applicable to a biomedical problem remains to be determined. Here we applied FISH-Flow to detection of latent Mycobacterium tuberculosis infection (LTBI), a condition affecting approximately a third of the world population. LTBI diagnosis is based on measuring IFN-γ release from peripheral blood mononuclear cells (PBMC) stimulated ex vivo with M. tuberculosis antigen. We obtained PBMC from 60 donors equally distributed between LTBI+ and LTBI- individuals, stimulated them for 6 hrs with M. tuberculosis purified protein derivative (PPD), and measured ex...
We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent ma... more We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent mammalian cells using fluorescence in situ hybridization (FISH) probes. The method, which we call FISH-Flow, allows for high-throughput multiparametric measurements of gene expression, a task that was not feasible with earlier, microscopy-based approaches. The FISH-Flow protocol involves cell fixation, permeabilization and hybridization with a set of fluorescently labeled oligonucleotide probes. In this protocol, surface and intracellular protein markers can also be stained with fluorescently labeled antibodies for simultaneous protein and mRNA measurement. Moreover, a semiautomated, single-tube version of the protocol can be performed with a commercially available cell-wash device that reduces cell loss, operator time and interoperator variability. It takes ∼30 h to perform this protocol. An example of FISH-Flow measurements of cytokine mRNA induction by ex vivo stimulation of primed T ce...
Fluorescence in situ hybridization coupled with flow cytometry (FISH-Flow) is a highly quantitati... more Fluorescence in situ hybridization coupled with flow cytometry (FISH-Flow) is a highly quantitative, high-throughput platform allowing precise quantification of total mRNA transcripts in single cells. In undiagnosed infections posing a significant health burden worldwide, such as latent tuberculosis or asymptomatic recurrent malaria, an important challenge is to develop accurate diagnostic tools. Antigen-specific T cells create a persistent memory to pathogens, making them useful for diagnosis of infection. Stimulation of memory response initiates T-cell transitions between functional states. Numerous studies have shown that changes in protein levels lag real-time T-cell transitions. However, analysis at the single-cell transcriptional level can determine the differences. FISH-Flow is a powerful tool with which to study the functional states of T-cell subsets and to identify the gene expression profiles of antigen-specific T cells during disease progression. Advances in instrumentat...
RNA flow cytometry (FISH-Flow) achieves high-throughput measurement of single-cell gene expressio... more RNA flow cytometry (FISH-Flow) achieves high-throughput measurement of single-cell gene expression by combining in-situ nucleic acid hybridization with flow cytometry. We tested whether antigen-specific T-cell responses detected by FISH-Flow correlated with latent tuberculosis infection (LTBI), a condition affecting one-third of the world population. Peripheral-blood mononuclear cells from donors, identified as positive or negative for LTBI by current medical practice, were stimulated ex vivo with mycobacterial antigen. IFNG and IL2 mRNA production was assayed by FISH-Flow. Concurrently, immunophenotypes of the cytokine mRNA-positive cells were characterized by conventional, antibody-based staining of cell-surface markers. An association was found between donor LTBI status and antigen-specific induction of IFNG and IL2 transcripts. Induction of these cytokine genes, which was detected by FISH-Flow in a quarter the time required to see release of the corresponding proteins by ELISA, ...
The use of monoclonal antibodies to distinguish human sarcoma from carcinoma cells has been explo... more The use of monoclonal antibodies to distinguish human sarcoma from carcinoma cells has been explored. Spleen cells from a BALB/c mouse immunized with a human malignant fibrohistiocytoma were fused with cells of the mouse P3U1 plasmacytoma cell line. Antibodies were then screened for reactivity against human sarcoma and carcinoma cells growing in culture. This work has yielded 2 immunoglobulin G monoclonal antibodies VIE4 and VIF3 which, respectively, reacted with 85% (17 of 20) and 90% (18 of 20) of sarcoma lines tested but with none of eight carcinoma cell line preparations. Reactivity against normal fibroblasts was also demonstrated. By immunofluorescence, the antigens detected by the two antibodies appear to have distinctive intracellular distributions. Immunoprecipitation with VIF3 has shown that it is detecting a protein with a molecular weight of 70,000. When tested against pathological frozen tissue sections, VIF3 reacted with four of 11 and VIE4 with three of 11 human sarcom...
A murine monoclonal antibody (MAb) was obtained that showed unique specificity for the immunizing... more A murine monoclonal antibody (MAb) was obtained that showed unique specificity for the immunizing T-cell line HPB-ALL. This antibody, C37 (an IgG,,K) also reacted with a small (2-5%) population of normal peripheral blood T (PBL-T) cells. These C37-positive (C37+) cells were found in both the T4/Leu3+ and T8/Leu2+ subsets. Like OKT3 antibody, C37 induced T-cell mitogenesis with a peak proliferative response at day 3. In longterm cultures containing irradiated autologous feeder cells and IL-2, C37 antibody caused the selective expansion of C37+ T cells. On HPB-ALL cells C37 induced comodulation of the T3 molecule. C37 precipitated a disulfide-linked dimer characteristic of the T-cell antigen receptor consisting of an a-subunit (45-48 kD) and a ß-subunit (38-42 kD) from both C37+ T-cell blasts of a normal individual and HPB-ALL cells that were surface radioiodinated. However, the precipitated molecule isolated from C37 antibody-activated T-cell blasts exhibited a different pi from that isolated from HPB-ALL cells. Our studies indicate that C37 recognizes an epitope on the T-cell receptor molecule that is shared by a subpopulation of human T cells, which raises the possibility that multiple variable-region associated and/or framework-like determinants of the T-cell antigen receptor can be defined serologically and used in functional and molecular studies of T-cell subsets.
Host responses to Mycobacterium tuberculosis infection provide a basis for diagnosis of latent in... more Host responses to Mycobacterium tuberculosis infection provide a basis for diagnosis of latent infection and active disease. T cell responses have been the mainstay for diagnosis of latent infection and may contribute to diagnosis of active disease. Recent advances in characterizing humoral responses will likely contribute to improved diagnosis of active disease. However, these measures fail to distinguish the continuum of infection states. Moving to a systems approach to biomarker discovery may provide the resolution that current methods of diagnosis lack. The chapter evaluates the current use of T cell and B cell responses for diagnosis and the limitations of applying them separately. The possibility that macrophage or monocyte activation may serve as a biomarker is also addressed. We consider whether methodologies that combine (a) multifunctional T cell responses and T cell types, (b) monocyte/macrophage characteristics that reveal response to infection, and (c) dominant B cell responses to M. tuberculosis growth-phase-specific antigens can further contribute to a systems approach for biomarker discovery that can distinguish among infection states.
We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent ma... more We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent mammalian cells using fluorescence in situ hybridization (FISH) probes. The method, which we call FISH-Flow, allows for high-throughput multiparametric measurements of gene expression, a task that was not feasible with earlier, microscopy-based approaches. The FISH-Flow protocol involves cell fixation, permeabilization and hybridization with a set of fluorescently labeled oligonucleotide probes. In this protocol, surface and intracellular protein markers can also be stained with fluorescently labeled antibodies for simultaneous protein and mRNA measurement. Moreover, a semiautomated, single-tube version of the protocol can be performed with a commercially available cell-wash device that reduces cell loss, operator time and interoperator variability. It takes ~30 h to perform this protocol. An example of FISH-Flow measurements of cytokine mRNA induction by ex vivo stimulation of primed T cells with specific antigens is described.
Host responses to Mycobacterium tuberculosis infection provide a basis for diagnosis of latent in... more Host responses to Mycobacterium tuberculosis infection provide a basis for diagnosis of latent infection and active disease. T cell responses have been the mainstay for diagnosis of latent infection and may contribute to diagnosis of active disease. Recent advances in characterizing humoral responses will likely contribute to improved diagnosis of active disease. However, these measures fail to distinguish the continuum of infection states. Moving to a systems approach to biomarker discovery may provide the resolution that current methods of diagnosis lack. The chapter evaluates the current use of T cell and B cell responses for diagnosis and the limitations of applying them separately. The possibility that macrophage or monocyte activation may serve as a biomarker is also addressed. We consider whether methodologies that combine (a) multifunctional T cell responses and T cell types, (b) monocyte/macrophage characteristics that reveal response to infection, and (c) dominant B cell responses to M. tuberculosis growth-phase-specific antigens can further contribute to a systems approach for biomarker discovery that can distinguish among infection states.
Flow cytometric characterization of Ag-specific T cells typically relies on detection of protein ... more Flow cytometric characterization of Ag-specific T cells typically relies on detection of protein analytes. Shifting the analysis to detection of RNA would provide several significant advantages, which we illustrate by developing a new host immunity-based platform for detection of infections. Cytokine mRNAs synthesized in response to ex vivo stimulation with pathogen-specific Ags are detected in T cells with single-molecule fluorescence in situ hybridization followed by flow cytometry. Background from preexisting in vivo analytes is lower for RNAs than for proteins, allowing greater sensitivity for detection of low-frequency cells. Moreover, mRNA analysis reveals kinetic differences in cytokine expression that are not apparent at the protein level but provide novel insights into gene expression programs expected to define different T cell subsets. The utility of probing immunological memory of infections is demonstrated by detecting T cells that recognize mycobacterial and viral Ags in donors exposed to the respective pathogens.
Fluorescence in situ hybridization coupled with flow cytometry (FISH-Flow) is a highly quantitati... more Fluorescence in situ hybridization coupled with flow cytometry (FISH-Flow) is a highly quantitative, high-throughput platform allowing precise quantification of total mRNA transcripts in single cells. In undiagnosed infections posing a significant health burden worldwide, such as latent tuberculosis or asymptomatic recurrent malaria, an important challenge is to develop accurate diagnostic tools. Antigen-specific T cells create a persistent memory to pathogens, making them useful for diagnosis of infection. Stimulation of memory response initiates T-cell transitions between functional states. Numerous studies have shown that changes in protein levels lag real-time T-cell transitions. However, analysis at the single-cell transcriptional level can determine the differences. FISH-Flow is a powerful tool with which to study the functional states of T-cell subsets and to identify the gene expression profiles of antigen-specific T cells during disease progression. Advances in instrumentation, fluorophores, and FISH methodologies will broaden and deepen the use of FISH-Flow, changing the immunological field by allowing determination of functional immune signatures at the mRNA level and the development of new diagnostic tools.
We have identified and characterized the tissue distribution of the antigen recognized by a novel... more We have identified and characterized the tissue distribution of the antigen recognized by a novel monoclonal antibody (mAb) IBI0, raised against an activated yo T cell clone. Immunohistochemistry of tissue sections, and analysis of single cell suspensions by flow cytometry revealed that mAb IBI0 weakly reacted with <6% of normal human peripheral blood mononuclear cells (PBMC). After 5-6 days of in vitro culture of PBMC activated with phytohemagglutinin (PHA), 55% of the CD4+ and 25% of the CD8+ T cells became 1B1O+. IBI0 expression was maintained on long term cultured interleukin 2 (IL-2)-dependent T cell receptor (TCR) aW and yo+ clones, and importantly, in contrast to resting T cells, the majority of in vivo activated synovial T lymphocytes from a patient with rheumatoid arthritis were IBI0+. In addition, myelo-monocytic U927 cells, tissue macrophages and some epithelia and fibroblasts were found to react with mAb IBIO. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of molecules immuno-precipitated by mAb IBIO from radio-iodinated cell surface membrane lysates of T lymphocyte and U937 cells revealed 26 and 29 kiloDalton (kDa) glycoproteins respectively. In conclusion, mAb I BI a recognizes a novel «late» appearing 26 kDa T cell activation antigen that may be useful for further studies of activated T cells in health and disease. Abbreviations: FCS =fetal calf serum; FM =final medium; FIYC =fluoresceine isothiocyanate; LBC =lymphoblastoid B cells; IL =interleukin-2; IL-2R =IL-receptor; Ig =immunoglobulin; IV = international units; IMDM = Iscoves modified Dulbecco medium; kDa = kilodalton; MHC = major histo compatibility; mAb = monoclonal antibody; PBMC = peripheral blood mononuclear cells; pmst = phenyl methyl sulfonyl fluoride; PBS = phosphate buffered saline; R IL-2 = recombinant IL-2; SDS-PAGE = sodium dodecyl sulfate polyacrilamide gel electrophoresis; SRBC =sheep red blood cell; YCR =T cell receptor; YBS =tris buffered saline; RA = rheumatoid arthritis; VLA =very late antigen.°1 999 by URBAN & FISCHER
Establishing diagnosis of latent and active histoplasmosis is challenging. Interferon gamma-relea... more Establishing diagnosis of latent and active histoplasmosis is challenging. Interferon gamma-release assays (IGRAs) may provide evidence of latent and active infection.
Cross-linking of CD8 and HLA class I molecules with appropriate monoclonal antibodies (mAb) and g... more Cross-linking of CD8 and HLA class I molecules with appropriate monoclonal antibodies (mAb) and goat anti-mouse Ig (GaMlg) antibody resulted in a marked proliferation of resting human CD8 cells in the presence of interleukin-2 (IL-2). These cells also expressed IL-2 receptor (IL-2R), transferrin receptor, HLA-DR and-DQ antigens. Activation of the cross-linked CD8 cells is apparently independent of accessory monocytes. Various anti-CD8 and anti-HLA class I mAb recognizing nonpolymorphic antigenic determinants were examined for the efficacy of activating CD8 cells. Among mAb specific for HLA class I molecules, PA2.6, MB40.5, BB7.7, A 1.4, and W6/32 mAb markedly stimulated the proliferation of cross-linked CD8 cells, whereas BBM. 1, Ql/28, and HClO mAb were found inactive. Footprinting analysis of HLA class 1 molecules suggested that the activity of these anti-HLA class I mAb appeared to be related to the corresponding peptides they protect from enzymatic digestion. In contrast to the anti-HLA class I mAb, all anti-CD8 mAb examined (C8, OKT8A, and anti-Leu-2a) induced the proliferation of CDS-HLA class I cross-linked cells with similar efficacy. These results suggest that physical interaction between CD8 and at least one specific region of HLA class I molecules can trigger the activation of resting human CD8 cells.
Five monoclonal antibodies (A7, B24, I14, L12, and M2) recognizing different epitopes of the huma... more Five monoclonal antibodies (A7, B24, I14, L12, and M2) recognizing different epitopes of the human natural IFN-γ were prepared by immunizing BALB/c mice with a highly purified human natural IFN-γ preparation (107 U/mg). All five antibodies had high IFN-γ-binding activity but exhibited differential IFN-γ-neutralizing activities. Furthermore, none of them neutralized the antiviral activity exhibited by either IFN-α or IFN-β preparations, indicating thus their specificity for IFN-γ. The A7, L12, M2, and I14 monoclonal antibodies, but not the B24, blocked the augmentation of natural killer cytotoxicity, mediated by peripheral blood monocyte-depleted lymphocytes, by Escherichia coli-derived IFN-γ or natural IFN-γ but not by IFN-α2. All five monoclonal antibodies precipitated an identical molecular complex containing two major protein components with molecular weights of 20,000 (20 kD) and 25,000 (25 kD) and two minor components with molecular weights of 17,000 (17 kD) and 45,000 (45 kD). Treatment of the immun...
A murine monoclonal antibody (MAb) was obtained that showed unique specificity for the immunizing... more A murine monoclonal antibody (MAb) was obtained that showed unique specificity for the immunizing T-cell line HPB-ALL. This antibody, C37 (an IgG,,K) also reacted with a small (2-5%) population of normal peripheral blood T (PBL-T) cells. These C37-positive (C37+) cells were found in both the T4/Leu3+ and T8/Leu2+ subsets. Like OKT3 antibody, C37 induced T-cell mitogenesis with a peak proliferative response at day 3. In longterm cultures containing irradiated autologous feeder cells and IL-2, C37 antibody caused the selective expansion of C37+ T cells. On HPB-ALL cells C37 induced comodulation of the T3 molecule. C37 precipitated a disulfide-linked dimer characteristic of the T-cell antigen receptor consisting of an a-subunit (45-48 kD) and a ß-subunit (38-42 kD) from both C37+ T-cell blasts of a normal individual and HPB-ALL cells that were surface radioiodinated. However, the precipitated molecule isolated from C37 antibody-activated T-cell blasts exhibited a different pi from that isolated from HPB-ALL cells. Our studies indicate that C37 recognizes an epitope on the T-cell receptor molecule that is shared by a subpopulation of human T cells, which raises the possibility that multiple variable-region associated and/or framework-like determinants of the T-cell antigen receptor can be defined serologically and used in functional and molecular studies of T-cell subsets.
A T cell membrane antigen-recognition structure has recently been described ill several laborator... more A T cell membrane antigen-recognition structure has recently been described ill several laboratories, including our own (1-6). This molecule is a heterodimer of approximately 80-90 Kd, composed of two disulfide-linked chains. It fulfills many of the requirements for the T cell receptor for antigen, such as major histocompatibility complex-restricted antigen recognition (2-4). This molecule has been studied on murine cells (1, 2, 4), human T cell clones (3), and human T cell leukemia cells (5). The latter study, from this laboratory, described monoclonal antibodies reactive with idiotype-like determinants on a human T cell leukemia (5). Of two antibodies, one reacted with a private idiotype-like determinant and another cross-reacted with 1-2% of normal T cells by immunofluorescence. Both antibodies immunoprecipitated the same ~80 Kd disulfide-linked heterodimer from the membrane-iodinated leukemic cells, and this molecule comodulated with the T3 molecule. Thus, strong indirect evidence suggested that these monoclonal antibodies were recognizing a T cell antigen receptor. The presence of private idiotypic determinants and shared determinants on this molecule suggested a general structure similar to that of immunoglobulin molecules (7). This paper compares two T cell antiidiotypic antibodies each specifically reactive with their respective leukemia T cell clone. Immunoprecipitation with these antibodies revealed major differences between the two idiotype-bearing heterodimer molecules. In addition, proliferation studies in response to Sepharose-linked antiidiotypic antibody revealed marked differences between the two T cell leukemias. Materials and Methods Cell Preparations. Cells derived from the peripheral blood of a patient with Sezary syndrome, patient SU, and another patient with T celt chronic lymphocytic leukemia
We have previously described fluorescence in situ hybridization for mRNA detection combined with ... more We have previously described fluorescence in situ hybridization for mRNA detection combined with flow cytometry (FISH-Flow) as a novel method to detect gene expression at a single-cell level. Using mRNA as analyte in flow cytometry presents multiple, biological and technical advantages. Proof-of-principle experiments have also shown that FISH-Flow can detect rare events including antigen-specific T cell responses. Whether the method is applicable to a biomedical problem remains to be determined. Here we applied FISH-Flow to detection of latent Mycobacterium tuberculosis infection (LTBI), a condition affecting approximately a third of the world population. LTBI diagnosis is based on measuring IFN-γ release from peripheral blood mononuclear cells (PBMC) stimulated ex vivo with M. tuberculosis antigen. We obtained PBMC from 60 donors equally distributed between LTBI+ and LTBI- individuals, stimulated them for 6 hrs with M. tuberculosis purified protein derivative (PPD), and measured ex...
We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent ma... more We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent mammalian cells using fluorescence in situ hybridization (FISH) probes. The method, which we call FISH-Flow, allows for high-throughput multiparametric measurements of gene expression, a task that was not feasible with earlier, microscopy-based approaches. The FISH-Flow protocol involves cell fixation, permeabilization and hybridization with a set of fluorescently labeled oligonucleotide probes. In this protocol, surface and intracellular protein markers can also be stained with fluorescently labeled antibodies for simultaneous protein and mRNA measurement. Moreover, a semiautomated, single-tube version of the protocol can be performed with a commercially available cell-wash device that reduces cell loss, operator time and interoperator variability. It takes ∼30 h to perform this protocol. An example of FISH-Flow measurements of cytokine mRNA induction by ex vivo stimulation of primed T ce...
Fluorescence in situ hybridization coupled with flow cytometry (FISH-Flow) is a highly quantitati... more Fluorescence in situ hybridization coupled with flow cytometry (FISH-Flow) is a highly quantitative, high-throughput platform allowing precise quantification of total mRNA transcripts in single cells. In undiagnosed infections posing a significant health burden worldwide, such as latent tuberculosis or asymptomatic recurrent malaria, an important challenge is to develop accurate diagnostic tools. Antigen-specific T cells create a persistent memory to pathogens, making them useful for diagnosis of infection. Stimulation of memory response initiates T-cell transitions between functional states. Numerous studies have shown that changes in protein levels lag real-time T-cell transitions. However, analysis at the single-cell transcriptional level can determine the differences. FISH-Flow is a powerful tool with which to study the functional states of T-cell subsets and to identify the gene expression profiles of antigen-specific T cells during disease progression. Advances in instrumentat...
RNA flow cytometry (FISH-Flow) achieves high-throughput measurement of single-cell gene expressio... more RNA flow cytometry (FISH-Flow) achieves high-throughput measurement of single-cell gene expression by combining in-situ nucleic acid hybridization with flow cytometry. We tested whether antigen-specific T-cell responses detected by FISH-Flow correlated with latent tuberculosis infection (LTBI), a condition affecting one-third of the world population. Peripheral-blood mononuclear cells from donors, identified as positive or negative for LTBI by current medical practice, were stimulated ex vivo with mycobacterial antigen. IFNG and IL2 mRNA production was assayed by FISH-Flow. Concurrently, immunophenotypes of the cytokine mRNA-positive cells were characterized by conventional, antibody-based staining of cell-surface markers. An association was found between donor LTBI status and antigen-specific induction of IFNG and IL2 transcripts. Induction of these cytokine genes, which was detected by FISH-Flow in a quarter the time required to see release of the corresponding proteins by ELISA, ...
The use of monoclonal antibodies to distinguish human sarcoma from carcinoma cells has been explo... more The use of monoclonal antibodies to distinguish human sarcoma from carcinoma cells has been explored. Spleen cells from a BALB/c mouse immunized with a human malignant fibrohistiocytoma were fused with cells of the mouse P3U1 plasmacytoma cell line. Antibodies were then screened for reactivity against human sarcoma and carcinoma cells growing in culture. This work has yielded 2 immunoglobulin G monoclonal antibodies VIE4 and VIF3 which, respectively, reacted with 85% (17 of 20) and 90% (18 of 20) of sarcoma lines tested but with none of eight carcinoma cell line preparations. Reactivity against normal fibroblasts was also demonstrated. By immunofluorescence, the antigens detected by the two antibodies appear to have distinctive intracellular distributions. Immunoprecipitation with VIF3 has shown that it is detecting a protein with a molecular weight of 70,000. When tested against pathological frozen tissue sections, VIF3 reacted with four of 11 and VIE4 with three of 11 human sarcom...
A murine monoclonal antibody (MAb) was obtained that showed unique specificity for the immunizing... more A murine monoclonal antibody (MAb) was obtained that showed unique specificity for the immunizing T-cell line HPB-ALL. This antibody, C37 (an IgG,,K) also reacted with a small (2-5%) population of normal peripheral blood T (PBL-T) cells. These C37-positive (C37+) cells were found in both the T4/Leu3+ and T8/Leu2+ subsets. Like OKT3 antibody, C37 induced T-cell mitogenesis with a peak proliferative response at day 3. In longterm cultures containing irradiated autologous feeder cells and IL-2, C37 antibody caused the selective expansion of C37+ T cells. On HPB-ALL cells C37 induced comodulation of the T3 molecule. C37 precipitated a disulfide-linked dimer characteristic of the T-cell antigen receptor consisting of an a-subunit (45-48 kD) and a ß-subunit (38-42 kD) from both C37+ T-cell blasts of a normal individual and HPB-ALL cells that were surface radioiodinated. However, the precipitated molecule isolated from C37 antibody-activated T-cell blasts exhibited a different pi from that isolated from HPB-ALL cells. Our studies indicate that C37 recognizes an epitope on the T-cell receptor molecule that is shared by a subpopulation of human T cells, which raises the possibility that multiple variable-region associated and/or framework-like determinants of the T-cell antigen receptor can be defined serologically and used in functional and molecular studies of T-cell subsets.
Host responses to Mycobacterium tuberculosis infection provide a basis for diagnosis of latent in... more Host responses to Mycobacterium tuberculosis infection provide a basis for diagnosis of latent infection and active disease. T cell responses have been the mainstay for diagnosis of latent infection and may contribute to diagnosis of active disease. Recent advances in characterizing humoral responses will likely contribute to improved diagnosis of active disease. However, these measures fail to distinguish the continuum of infection states. Moving to a systems approach to biomarker discovery may provide the resolution that current methods of diagnosis lack. The chapter evaluates the current use of T cell and B cell responses for diagnosis and the limitations of applying them separately. The possibility that macrophage or monocyte activation may serve as a biomarker is also addressed. We consider whether methodologies that combine (a) multifunctional T cell responses and T cell types, (b) monocyte/macrophage characteristics that reveal response to infection, and (c) dominant B cell responses to M. tuberculosis growth-phase-specific antigens can further contribute to a systems approach for biomarker discovery that can distinguish among infection states.
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Papers by Yuri Bushkin