Establishment and characterization of a chicken mononuclear cell line

Background

Flow cytometric fluorescence resonance energy transfer (FCET) is an efficient method to map associations between biomolecules because of its high sensitivity to changes in molecular distances in the range of 1–10 nm. However, the requirement for a dual-laser instrument and the need for a relatively high signal-to-noise system (i.e., high expression level of the molecules) pose limitations to a wide application of the method.



Methods

Antibodies conjugated to cyanines 3 and 5 (Cy3 and Cy5) were used to label membrane proteins on the cell surface. FCET measurements were made on a widely used benchtop dual-laser flow cytometer, the FACSCalibur, by using cell-by-cell analysis of energy transfer efficiency.



Results

To increase the accuracy of FCET measurements, we applied a long wavelength donor–acceptor pair, Cy3 and Cy5, which beneficially affected the signal-to-noise ratio in comparison with the classic pair of fluorescein and rhodamine. A new algorithm for cell-by-cell correction of autofluorescence further improved the sensitivity of the technique; cell subpopulations with only slightly different FCET efficiencies could be identified. The new FCET technique was tested on various direct and indirect immunofluorescent labeling strategies. The highest FCET values could be measured when applying direct labeling on both (donor and acceptor) sides. Upon increasing the complexity of the labeling scheme by introducing secondary antibodies, we detected a decrease in the energy transfer efficiency.



Conclusions

We developed a new FCET protocol by applying long wavelength excitation and detection of fluorescence and by refining autofluorescence correction. The increased accuracy of the new method makes cells with low receptor expression amenable to FCET investigation, and the new approach can be implemented easily on a commercially available dual-laser flow cytometer, such as a FACSCalibur. Cytometry 48:124–135, 2002. © 2002 Wiley-Liss, Inc.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/cyto.10121

Long wavelength fluorophores and cell-by-cell correction for autofluorescence significantly improves the accuracy of flow cytometric energy transfer measurements on a dual-laser benchtop flow cytometer

Background: Spinal cord injury (SCI) is a widely spread pathology with currently no effective treatment for any symptom. Regenerative medicine through cell transplantation is a very attractive strategy and may be used in different non-exclusive ways to promote functional recovery. We investigated functional and structural outcomes after grafting human embryonic neural progenitors (hENPs) in spinal cord-lesioned rats. Methods and Principal Findings: With the objective of translation to clinics we have chosen a paradigm of delayed grafting, i.e., one week after lesion, in a severe model of spinal cord compression in adult rats. hENPs were either nao ¨ve or engineered to express Neurogenin 2 (Ngn2). Moreover, we have compared integrating and non-integrating lentiviral vectors, since the latter present reduced risks of insertional mutagenesis. We show that transplantation of hENPs transduced to express Ngn2 fully restore weight support and improve functional motor recovery after severe spinal cord compression at thoracic level. This was correlated with partial restoration of serotonin innervations at lumbar level, and translocation of 5HT1A receptors to the plasma membrane of motoneurons. Since hENPs were not detectable 4 weeks after grafting, transitory expression of Ngn2 appears sufficient to achieve motor recovery and to permit axonal regeneration. Importantly, we also demonstrate that transplantation of nao ¨ve hENPs is detrimental to functional recovery. Conclusions and Significance: Transplantation and short-term survival of Ngn2-expressing hENPs restore weight support after SCI and partially restore serotonin fibers density and 5HT1A receptor pattern caudal to the lesion. Moreover, grafting of nao ¨ve-hENPs was found to worsen the outcome versus injured only animals, thus pointing to the possible detrimental effect of stem cell-based therapy per se in SCI. This is of major importance given the increasing number of clinical trials involving cell grafting developed for SCI patients.

/pdf/grafted-human-embryonic-progenitors-expressing-neurogenin-2-9kr3ov7gem.pdf

Grafted human embryonic progenitors expressing neurogenin-2 stimulate axonal sprouting and improve motor recovery after severe spinal cord injury.

In this chapter we would like to review evidence supporting the concept that there is extensive heterogeneity of mammalian mononuclear phagocytes. Given the limits of this chapter, we will summarize data supporting this notion rather than provide an in-depth review of particular characteristics and functions of these cells. Therefore, the main subjects will be the mononuclear phagocyte system as a whole and the heterogeneous features of the various members of the system expressed both phenotypically and functionally. Finally, we will discuss various models that may explain how mononuclear phagocyte heterogeneity is generated (see also Chapter 3, this volume).

Heterogeneity of Mononuclear Phagocytes

Anti-idiotypic antibodies (Ab2) that functionally mimic epitopes associated with human cancer cells are the most specific cancer vaccines currently available. Ab2 can induce specific humoral anti-tumor immunity in cancer patients. However, the potential of Ab2 for inducing cellular immunity in cancer patients still requires demonstration. Clonotypic antibodies directed against the combining site for tumor Ag on human T-cell clones may provide highly effective reagents for inducing protective T-cell immunity against human cancer. A new generation of cancer vaccines, molecularly cloned tumor-associated antigens (Ag), has recently been developed. Recombinant Ag have been successfully expressed in vectors allowing large scale production of Ag for immunization of cancer patients. Recombinant tumor Ag was shown to induce specific and protective immunity in experimental animals. In contrast to Ab2, which may mimic a single cancer-associated epitope, recombinant Ag express multiple epitopes that are potentially immunogenic. Ag vaccines, therefore, may be more effective in arresting tumor growth than single epitope (Ab2) vaccines because tumor destruction by antibodies is dependent on antibody density on tumor cell surfaces. In light of the important roles that both B and T cells play in the control of tumor growth, the demonstration of induction of specific B and T cell-immunity by recombinant tumor Ag and Ab2 in experimental animals is encouraging. Ultimately, the immunomodulatory role of both types of vaccines has to be compared in cancer patients who are immunologically tolerant to many Ag/epitopes expressed by their growing tumors. The development of both Ab2 and recombinant Ag for single antigenic systems provides the first step towards this goal.

Epitope- and Antigen-specific Cancer Vaccines

Attempts to analyze bone marrow-derived macrophages (BMDM) by flow cytometry have been prohibited because of their high autofluorescence. Using an autofluorescence reduction method of Steinkamp and Stewart to reduce the autofluorescence of BMDM, we were able to examine several macrophage populations for their expression of I-A, I-J, and Mac-1 cell surface determinants. Bone marrow cells examined immediately after removal from the femur contain 50-60% Mac 1-positive cells (mainly granulocytes). During the next few days granulocytes and nonmacrophage precursor cells die, and the number of Mac 1-positive cells decrease. Once the bone marrow cells have been maintained in L cell conditioned medium (LCM) for 2 to 3 days, the number of cells expressing Mac 1 increases rapidly from 20% to 98% during the next 3 to 4 days. Bone marrow cells grown in LCM do not express I-J until these cells have been in culture 3 to 4 days, and the number of cells expressing I-J (up to 90% positive) parallels the increase in macrophages. Bone marrow cells maintained in LCM did not express detectable I-A during the 14 days these cells were examined. Two other macrophage populations often used in a variety of immunological studies were analyzed by flow cytometry. We found that the majority (up to 80%) of peritoneal cells expressed I-A, and only 20% of peritoneal cells had I-J cell surface determinants. On the other hand, peritoneal exudate cells collected 4 days after thioglycolate medium treatment were predominantly I-J positive (up to 70%), and only about 30% of these cells expressed I-A cell surface antigens. The binding of anti-I-J IgM antibody to BMDM was not to Fc receptors because pretreating these cells with up to 25 micrograms of an IgG2a myeloma protein did not block anti-I-J antibody binding. The addition of 25-200 micrograms of monoclonal anti-Fc receptor antibody was also ineffective in blocking the binding of a monoclonal anti-I-Jk antibody to BMDM. Pretreatment of BMDM with the IgM fraction of several control IgM antibody preparations did not block the specific binding of fluoresceinated anti-I-J IgM antibody. BMDM provide a pure population of macrophages that express a significant level of cell surface I-J antigens. Bone marrow cells grown in LCM are essentially devoid of other contaminating cells, and the increase in the number of I-J-positive cells parallels the increase in macrophages in these cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow cytometric analysis of I-J expression on murine bone marrow-derived macrophages.

Cultures of normal spleen cells with anti-idiotypic antibody (anti-Id) or antigen B (AgB)-specific T suppressor factor (Tsf1) in mini-Marbrook chambers for 4 days at 37 degrees C lead to the in vitro induction of AgB-specific T suppressor (TS) cells. These TS cells significantly suppress a secondary AgB-specific IgE response, but they do not affect a secondary AgB-specific IgG response. Depletion of both B cells and macrophages from normal spleen cells by panning on anti-Ig-coated petri dishes provides an enriched T cell population. These enriched T cells when cultured with anti-Id or Tsf1 in mini-Marbrook chambers do not produce AgB-specific TS cells, and mice treated with cells harvested from the mini-Marbrook chambers have normal secondary AgB-specific IgG and IgE responses. The addition of as few as 1000 bone marrow-derived macrophages (BMDM) to cultures of the enriched T cells with anti-Id, or Tsf1 restores the ability of these cultures to produce significant levels of AgB-specific TS cells. Further studies reveal that the macrophage population must be histocompatible and express a cell surface I-J antigen. Attempts to pulse BMDM with anti-Id or Tsf1 at 4 degrees C and to culture in mini-Marbrook chambers 10(3) pulsed BMDM with enriched T cells were unsuccessful in producing AgB-specific TS cells. However, pulsing BMDM with anti-Id or Tsf1 at 37 degrees C, and adding 10(3) of these pulsed BMDM to enriched T cells in culture led to the formation of significant levels of AgB-specific TS cells.

The role of macrophages in anti-idiotypic antibody and T suppressor factor induction of timothy grass pollen antigen B-specific T suppressor cells.

In this paper we describe production of alloantisera specific for determinants encoded by I-J gene loci expressed on macrophages. B10.A(3R) (I-Jb) mice were hyperimmunized with pure macrophages grown in vitro from bone marrow stem cells of congenic B10.A(5R) mice. The antisera contained predominantly IgM antibody that was non-adherent to protein-A-Sepharose with a minor component of IgG1, and IgG2a antibodies that were adherent to protein-A-Sepharose. The protein-A non-adherent antibody completely blocked the in vitro generation of humoral immune responses to sheep erythrocytes by spleen cell from B10.A(5R) mice and from inbred strains that share the I-Jk haplotypes, but did not alter the responses of spleen cells of the I-Jb haplotype. In the presence of complement, both protein-A adherent and protein-A non-adherent antibodies eliminated the capacity of B10.A(5R) spleen cells to generate humoral and proliferative responses, but the functional activity of B10.A(3R) cells was unaffected. These data indicate the I-Jk specificity of the antisera. The capacity of the anti-macrophage antibody to block humoral immune induction was removed by absorption with bone marrow-derived macrophages from B10.A(5R) mice, but not from B10.A(3R) mice. Further, the B10.A(5R) macrophages completely restored the humoral responses of antibody- and complement-treated B10.A(5R) spleen cells, but B10.A(3R) macrophages showed only partial restoration that was consistent with a factor-mediated allogeneic effect. These data demonstrate the specificity of our anti-I-J sera for macrophages and indicate that bone marrow-derived macrophages express surface I-J encoded molecules.

Anti-I-J alloantisera elicited by immunization of B10.A(3R) (I-Jb) mice with bone marrow-derived macrophages from B10.A(5R) (I-Jk) mice.

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Flow cytometric analysis of I-J expression on murine bone marrow-derived macrophages.

The role of macrophages in anti-idiotypic antibody and T suppressor factor induction of timothy grass pollen antigen B-specific T suppressor cells.

Anti-I-J alloantisera elicited by immunization of B10.A(3R) (I-Jb) mice with bone marrow-derived macrophages from B10.A(5R) (I-Jk) mice.