O

Coll at Interest

Cell bibclcd with brUinylnrod antibody

Cell bibclcd with brUinylnrod antibody

Biol in libeled wilh flvidin bono»

Biol in libeled wilh flvidin bono»

Liiboloi! colli retained m mngneli; (lew

H6l«J.l«« 01 Libeled tolls by removkig Titiq rwL

Figure 1 Schematic representation of the use of paramagnetic beads for 'positive' enrichment. (A) The desired cells are labeled with a biotinylated antibody, washed, then attached to avidin beads. (B) Labeled cells are retained when a magnetic field is applied, and unwanted cells washed through. The desired cells are then recovered after removal of the column or tube from the magnetic field.

slower and more accurate sort is reduced resulting in an overall saving of time.

Isolation: fluorescence-activated cell sorting

FACS provides the most reliable and accurate means of purifying antigen-specific B cells. B cells can be identified by a number of markers, but the ability to detect binding of a specific antigen is crucial to identifying antigen-specific cells. Initial success using this method was achieved by Hayakawa and colleagues, who immunized mice with the fluorescent protein phycoerythrin (PE), later isolating PE-bind-ing B cells on the FACS. This technique was later advanced by Lalor, McHeyzer-Williams and Nossal, who identified NP-specific B cells by detecting binding of NP conjugated to allophycocyanin, allowing further dissection of a well-characterized stereotypic immune response.

A major drawback of this technique has been the time (and therefore expense) taken to sort substantial number of cells if these are very rare. This can now be largely overcome by using one or a combination of the pre-enrichment techniques described above.

Another advance has been the advent of techniques allowing analysis of single cells, which removes the need to sort large numbers of cells. For example, a polymerase chain reaction (PCR)-based strategy can be used to amplify single genes from cDNA which can subsequently be sequenced. This has allowed analysis of patterns of somatic hypermutation in very rare subpopulations of antigen-specific B cells, such as the bone marrow antibody-forming cells described below.

Another major problem is the potential difficulty in ensuring the purity of very rare populations of cells, as relatively few cells which are incorrectly sorted can reduce the purity of such populations dramatically. This has been overcome by making maximal use of the number of detection parameters available on modern FACS machines. Consider the example of the isolation of NP-specific bone marrow antibody-forming cells shown in Figure 2. First, all cells which do not have the forward and side scatter characteristics (which measure size and 'complexity' respectively) of lymphocytes are excluded. One channel is then used to identify and exclude unwanted

Syndecan ^ NP-Binding -

Figure 2 Identification of NP-specific antibody-forming cells in the bone marrow after 14 days of the primary immune response. (A) Cells staining for IgM, IgD and Gr-1 and propidium iodide were excluded. Antibody-forming cells were identified by their expression of syndecan. (B) This population was further partitioned into cells expressing lgG1 and able to bind the immunizing hapten NP, coupled to a fluorochrome. In the example shown, the NP-specific antibody-forming cell population represents 0.003% of nucleated bone marrow cells. Boxes indicate the criteria used for sorting cells for VH gene sequencing.

Syndecan ^ NP-Binding -

Figure 2 Identification of NP-specific antibody-forming cells in the bone marrow after 14 days of the primary immune response. (A) Cells staining for IgM, IgD and Gr-1 and propidium iodide were excluded. Antibody-forming cells were identified by their expression of syndecan. (B) This population was further partitioned into cells expressing lgG1 and able to bind the immunizing hapten NP, coupled to a fluorochrome. In the example shown, the NP-specific antibody-forming cell population represents 0.003% of nucleated bone marrow cells. Boxes indicate the criteria used for sorting cells for VH gene sequencing.

cells. Antibodies to IgM, IgD and the myeloid-macrophage lineage marker Gr-1 are conjugated to PE, and dead cells are labeled with propidium iodide (which fluoresces in the PE channel), and all of the cells so labeled can be excluded from further analysis (a 'dump' channel). The addition of a dump channel to a single positive selection channel can decrease nonspecific contamination to less than 1 in 106 cells. Two further antibodies identify markers characteristic of the cells required - syndecan (a marker of antibody-forming cells, in the fluorescein channel) and surface IgGl (in the Texas Red channel). Finally a sixth parameter is used to ensure antigen specificity, that is, the ability of the IgM" IgD " Gr-1" syndecan+ IgGl+ cells to bind the original immunizing antigen NP bound to allophycocyanin. The probability of a nonspecifically staining cell having the same characteristics as the required cells in all six parameters is so low that populations of antigen-specific cells as rare as 1 in 200 000 cells can now be reliably and consistently sorted to >95% purity, even in the absence of pre-enrichment (see Figure 2).

Isolation: histology

The groups of Kelsoe, Rajewsky and others have recently isolated antigen-specific B (and germinal center T) cells by micromanipulation of single nuclei from histological sections. While this can only isolate small numbers of nuclei for PCR analysis, it does permit correlation of the molecular information obtained by PCR with the exact location of the cell in a stained histological section. This has allowed elegant analyses to be performed of the development of the postantigenic B cell response which would not have been possible using conventional techniques.

Isolation: transgenic mice

Another approach to isolation of antigen-specific cells is the creation of mice bearing transgene-enco-ded antigen receptors of known specificity. Well-known examples are the mice transgenic for a T cell receptor specific for the male HY antigen, and those transgenic for immunoglobulin binding hen egg lyso-zyme. Thus the vast majority of the lymphocytes in such mice are of known specificity. This avoids the need to isolate rare cells, but requires careful interpretation of results since they have been obtained from mice with a markedly skewed lymphocyte repertoire.

How To Bolster Your Immune System

How To Bolster Your Immune System

All Natural Immune Boosters Proven To Fight Infection, Disease And More. Discover A Natural, Safe Effective Way To Boost Your Immune System Using Ingredients From Your Kitchen Cupboard. The only common sense, no holds barred guide to hit the market today no gimmicks, no pills, just old fashioned common sense remedies to cure colds, influenza, viral infections and more.

Get My Free Audio Book


Post a comment