Alternative Antigen Transport Pathway Chart


Ag, antigen; VC, veiled cell; SS, subcapsular sinus of lymph node; ATC, antigen transport cells; pre FDC, FDC precursor; FDC, follicular dendritic cells; ICCOSOMES, immune complex coated bodies; Bm, B memory cells; Th, T helper cells; GC, germinal center; MEDULLA, lymph node medulla; BM, bone marrow; Pc, plasma cell; Ab, antibody.

This chart shows the current working model for the:
1. TRANSPORT of antigen from the subcapsular sinus of a lymph node to the lymphoid nodule follicle);
2. The PRESENTATION of antigen by the B cell to T helper cells;
3. The rout to germinal center formation and antibody production;
4. The FEEDBACK regulation of the anamnestic humoral immune response.

The following is a brief interpretation of this model with reference to the chart. To see actual electron micrographs or data, scroll down for references to figures in the chart in succession. Please note that in most biological systems redundancies exist. Similarly, in antigen transport, other transport mechanism may exist which may operate in alternative ways. Another antigen transport mechanism is said to involve antigen transport by lymphocytes. Both this alternative antigen transport pathway and a transport mechanism involving lymphocytes are discussed in detail in the book (CTMI, 201) edited by Dr. Kosco-Vilbois. These are references by: Heinen et al., and by Szakal et al. [REF1] [REF2]
The IMMUNOLOGICAL EVENTS in the alternative antigen transport pathway of immune mice are as follows:

Sequence of events:

1 min-24 hrs
Ag-Ab-C' complexes are transported from the subcapsular sinus (SS) to follicles where the FDC-network (or reticulum) develops;
Day 1-3
Iccosome formation and dispersion of the immune complex coated beads or bodies (=somes) that are released by the FDC to transport the Ag to the B cells.
Day 3-5
Endocytosis of iccosomes, Ag processing / presentation to Th cells by Bm cells;
Day 3-10
Germinal center (GC) development.
Day 3-7
Ab-forming precursors in GC migrate to lymph node medulla and bone marrow to produce Ab.
Day 8-14
Ab production in medulla and bone marrow by plasma cells results in and the formation of a "ball of yarn"-like FDC dendrite convolutions for long-term retention of Ag.

With time, decrease in circulating Ab leads to dissociation of Ag-Ab-C' complexes, the ball-like dendrite convolutions unravel, expose the retained Ag and the FDCs are activated for a new cycle of iccosome production. This cycling is thought to help maintain Ab levels and long-term immunity.

Electron Micrographs and Data - Refer to Chart

Cell figure labeled VC:

An electron micrograph (EM) showing an antigen transport cell (ATC) in the subcapsular sinus (SS) of a popliteal lymph node (LN) 1 minute after subcutaneous introduction of the antigen into the foot of an immune animal. The popliteal LN drains the tissue fluid (lymph) from the foot region of the body. In this experiment the question of the transport of antigens [such as horseradish peroxidase (HRP)] to lymphoid nodules (follicles) in the lymph node cortex was studied. The antigen, HRP was identified in electron micrographs as a black precipitate detectable with a reaction for peroxidase. In this EM a relatively large cell is seen in the subcapsular sinus with the black antigenic material associated with its surface. The surface of this cell has "flap-like" veils. It is these veils that are coated with the antigen (black material). On the left side of the cell, these veils are freely visible and are coated with a granular black precipitate. On the rest of the surface of the cell because it is squeezed tightly into the SS, these veils are flattened against the cell body. These veils are also coated with the antigen. Thus, it may be said that this cell arrived to the lymph node SS in the lymph while transporting the antigen on its surface. This ATC was otherwise identified as a veiled-like cell. For more details on this study refer to this [REF].

Cell figure labeled ATC exiting SS:

An electron micrograph (EM) showing an antigen transport cell (ATC) in the process of passage through a pore in the floor (FL) of the subcapsular sinus (SS). This ATC passes into the cortex of a popliteal lymph node (LN) shortly after the subcutaneous introduction of antigen into the foot of an immune animal. The antigen in the form of immune complexes is bound to the surface of the process of this ATC. These immune complexes can be seen as black (electron dense) precipitate on the surface of veil-like processes which form a "catcher's-mitten" (P) that extends into the lumen of the SS. The veils coated with the Ag-Ab-C' complexes appear to be held together by the immune complexes. This kind of ATC processes can bee seen frequently in the act of being pulled through the pores in the floor of the SS. For more information consult our publication [REF].

Cell figure labeled pre FDC:

This color enhanced TEM shows the development of ATCs after passing through the floor of the subcapsular sinus. A non-colorized version of this EM was published in the original paper on antigen transport [REF]. The process (P1) of the cell labeled ATC is still being drawn through the pore of the SS floor. This processes extends to the cell body which possess an irregularly cleaved nucleus. The cell body also developed several additional processes which interdigitate with similar cell processes of the deeper lying FDCs. Judging from the lack of complicated convolutions, some of these cell processes may be more veil like then dendritic. These are antigen retaining cell processes as can be seen from the associated black immune complex deposits. Deeper in the cortex the processes of FDCs show a more dendritic pattern as suggested by the more convoluted appearance of the processes and immune complex deposits. The multi-cleaved nucleus is typical of early ATCs. It is believed that these lobes will completely separate giving rise to distinct nuclei without conventional karyokinezis. With maturation this would help form more FDCs. In support of this idea, it perhaps should be noted that FDCs could not be shown to replicate with conventional tritiated-Thdn labeling.

Cell figure labeled FDC (chart center):

This is a scanning electron micrograph (SEM) of an FDC isolated 1 Day after antigenic stimulation from mice. This FDC has filament-like (filiform) dendrites emanating from the central cell body. Lots of the dendrites have a club-shaped ending. Such club-shaped ends usually mean that the cell process is still growing. Some but not all FDCs become beaded to form iccosomes (see next figure of an FDC at 1-3 Day). Although it is not discernible on scanning electron micrographs, the dendrites and cell body are covered with a layer of immune complexes. Magnification: X3700. For more information refer to this [REF].

Cell figure labeled FDC D1-3:

This is a scanning electron micrograph (SEM) of an FDC isolated after antigenic stimulation from mice. The FDC was in the process of iccosome formation as can be seen from the numerous beads formed by its dendrites. Observe that near the origin of the dendrites the beads are imbedded in an amorphous material (probably immune complexes). Arrow: FDC cell body; L: lymphocyte. Mag.: X3700.
This and other observations [REF] suggested the idea that B cells acquire antigen by endocytosing dispersed iccosomes [REF]. For a demonstration of the coat of immune complexes on iccosomes click on the next event in the pathway, the dispersion of iccosomes.

Figure labeled Iccosome Dispersion:

Electron micrograph showing iccosome dispersion and endocytosis of iccosomes by germinal center B lymphocytes [REF]. The iccosomes are outlined by the black stained immune complexes. The black represents the reaction product formed at the site of the enzyme, horse radish peroxidase (or HRP), used as the antigen. The two iccosomes associated with the large B cell, which are indicated by two opposing arrows, are shown enlarged in the inset located in the left bottom corner. These iccosomes are surrounded by sheet-like veils for endocytosis. Most of the iccosomes are dispersed among the B cells of the forming germinal center. The upper left inset shows enlarged a group of iccosomes located intercellularly as indicated by the arrow heads. Iccosome dispersion and endocytosis take place typically around Day 3 after antigenic challenge. The result of the endocytosis of the iccosomal antigen is seen at the white arrow.
For further details the reader is referred to the original publication [REF].

Cell figure labeled D8-14:

An electron micrograph (EM) showing the convolutions of the FDC dendrities (FDC-D) located among several germinal center B lymphocytes. The convolutions seen represent a section through FDC dendrites outlined in this electron micrograph by the electron dense precipitate formed during the reaction of the antigen, HRP, with its substrate. This precipitate represent the location of HRP (or Ag)-Ab-C' complexes. The FDC dendrites are tightly balled up as the result of the binding of the immune complex on the surface of the dendrites to Fc (CD32) and C' receptors (CD21). This happens when antibody levels to the antigen rise (as immunity develops). After a time, when antibody levels drop in the circulation, antibody dissociates from these immune complexes and the dendrites are freed. This will again expose the antigen on the dendrites to the surrounding lymphocytes and the FDC can enter a new cycle in the alternative antigen transport pathway. For more information on this FDC control of the retained antigen see: [REF]. For the significance of this event consult: [REF].

Figure labeled Ab LEVEL DROPS and FEEDBACK:


This hypothesis is described in detail in the review article [REF] along with the experimental evidence leading to its development. Since 1980, new data was developed. For example the new observations on antigen transport and on the maturation of FDCs [i.e., from the filiform (yarn or string) type dendrites to the beaded dendrites), and the discovery of ICCOSOMEs. These have been since incorporated into the alternative antigen transport pathway along with the observation of the formation of the "ball of yarn" configuration of filiform FDC dendrites at the end of two weeks after antigen injection into immune mice.

FIGURE 1. A model for feedback regulation of antibody synthesis during the maintenance phase of the immune response. The injection of antigen and the subsequent induction of the primary immune response results in the production of specific antibody, the generation of a population of primed lymphocytes, and a population of follicular dendritic cells with surface associated persisting antigen. Later in the maintenance phase of the immune response, multiple dynamic equilibria are postulated to exist between persisting antigen, free specific antibody, and antigen- antibody complexes of various ratios in the lymphoid organs. The immunogenicity of the complexes is directly related to the antigen-antibody ratio and alterations in serum antibody levels result in formation or dissociation of these complexes. When antibody levels in the circulation decline, antigenic determinants are exposed, memory B- cells are stimulated, and a new cycle of antibody synthesis is induced. This newly produced antibody feeds back and terminates the immunogenic stimulus. Repetition of this cycle serves to maintain serum antibody levels within narrow limits for long periods of time. -- For further detailes of this review see: The Maintenance and Regulation of the Humoral Immune Response:Persisting antigen and the Role of Follicular Antigen-Binding Dendritic Cells. By Tew, Phipps and Mandel, Immunol.Rev. 1980, Vol. 53, pp. 175-201. [REF].

Figure labeled Pc D3-7:

This is an electron micrograph of a plasm a cell in the connective tissue of a horseradish peroxidase (HRP) immunized animal. A thick section was incubated in a solution of HRP to localize antigen-specific plasma cells. The rationale was based on reported observations that an antigen i.e., HRP, can bind to the anti-HRP antibodies in the endoplasmic cisternae of plasma cells the site of release of newly synthesized antibodies. The HRP is histo - chemically detectable by a t;substrate/DAB reaction and the precipitate forming at the site of HRP is electron dense (black). The black staining in the rough endoplasmic reticulum (RER) highlights the RER and indicates the presence of HRP-specific antibody. This method can be used for the detection of specific antibodies both histologically and by electron microscopy. [REF]

Cells figure labeled Presentation D-5:

This electron micrograph (EM) illustrates the uptake of iccosomal antigen at 5 days after injection of the antigen, HRP. By this time all iccosomes appear to have been endocytosed. The typical morphology of iccosomes is rapidly destroyed after ednocytosis and only the HRP antigen can be visualized histochemically in the cytoplasm of these germinal center B cells [See arrows indicating the electron dense (black) antigen. SEE LEFT EM]. Also, at this time the proliferative activity of these B cells becomes prominent. At higher magnification [SEE RIGHT EM] the path of the endocytosed antigen can be followed to the Golgi apparatus This high power EM on the right shows that the endocytosed antigen is transported in vesicles (black arrows). The vesicles migrate toward the Golgi located between the nucleus and the centriole and are indicated by the white arrows. Note that the antigen becomes associated with the Golgi membranes which typically function in the transport of newly formed membranes to the surface of the cell. The vesicles sent to the surface and probably contain the processed antigen for re-expression and presentation to helper T cells. For more detail on the morphology see the original paper: Refer to [REF].

Figure labeled Endocyt:

Electron micrographs showing the attachment and endocytosis of the iccosome at day 3 of the response. In A, an ICCOSOME, [Immune Complex COated SOME (=body)] is delivering antigen for edocytosis and processing and it is shown attached via the black HRP-anti HRP globules on its surface to a germinal center B lymphocyte. In B, an iccosome is being endocytosed (arrow) by a germinal center B cell. [REF]

Figure labeled Gc (Germinal Center):

An illustration of a lymphoid nodule (follicle) from a mouse spleen showing a well developed germinal center (GC). The germinal center is surrounded by its mantle (cortex) of hematoxilyn stained (light blue) lymphocytes. In the germinal center the Follicular Dendritic Cell (FDC) network is visualized histochemically using the monoclonal antibody, FDC-M1, in conjunction with biotin amplification and aminoethyl carbazole development. Note the crescent shaped magenta-colored FDC Network.