Mature Self Reactive B Cells Can Be Negatively Selected in the Periphery

Because some self-antigens do not have access to the bone marrow, B cells expressing mIgM specific for such antigens cannot be eliminated by the negative-selection process in the bone marrow described earlier. To avoid autoimmune responses from such mature self-reactive B cells, some process for deleting them or rendering them inactive must occur in peripheral lymphoid tissue.

A transgenic system developed by C. Goodnow and his coworkers has helped to clarify the process of negative selection of mature B cells in the periphery. Goodnow's experimental system included two groups of transgenic mice

Cell Cell Contact

FIGURE 11-11 Transmission electron micrographs of initial contact between a T cell and B cell (left) and of a T-B conjugate (right). Note the

broad area of membrane contact between the cells after formation of the conjugate. [From V. M. Sanders et al, 1986, J. Immunol. 137:2395.]

FIGURE 11-11 Transmission electron micrographs of initial contact between a T cell and B cell (left) and of a T-B conjugate (right). Note the

FIGURE 11-11

broad area of membrane contact between the cells after formation of the conjugate. [From V. M. Sanders et al, 1986, J. Immunol. 137:2395.]

Transgenic (HEL)

Transgenic (HEL)

Transgenic (anti-HEL)

Transgenic (anti-HEL)

Double transgenic (carrying both HEL and anti-HEL transgenes)

Double transgenic (carrying both HEL and anti-HEL transgenes)

Non-transgenic

Anti-HEL transgenic

Anti-HEL/HEL double transgenic

Non-transgenic

Anti-HEL transgenic

Anti-HEL/HEL double transgenic

10 100 1 10 100 1 10 100 IgM expression on membrane (arbitrary fluorescence units)

FIGURE 11-12

Goodnow's experimental system for demonstrating clonal anergy in mature peripheral B cells. (a) Production of double-trans-genic mice carrying transgenes encoding HEL (hen egg-white lysozyme) and anti-HEL antibody. (b) Flow cytometric analysis of peripheral B cells that bind HEL compared with membrane IgM levels. The number of B cells binding HEL was measured by determining how many cells bound fluorescently labeled HEL. Levels of membrane IgM were determined by incubating the cells with anti-mouse IgM antibody labeled with a fluorescent label different from that used to label HEL. Measurement of the flu orescence emitted from this label indicated the level of membrane IgM expressed by the B cells. The nontransgenics (left) had many B cells that expressed high levels of surface IgM but almost no B cells that bound HEL above the background level of 1. Both anti-HEL transgenics (middle) and anti-HEL/HEL double transgenics (right) had large numbers of B cells that bound HEL (blue), although the level of membrane IgM was about twentyfold lower in the double transgenics. The data in Table 11-3 indicate that the B cells expressing anti-HEL in the double transgenics cannot mount a humoral response to HEL.

(Figure 11-12a). One group carried a hen's egg-white lysozyme (HEL) transgene linked to a metallothionine promoter, which placed transcription of the HEL gene under the control of zinc levels. The other group of transgenic mice carried rearranged immunoglobulin heavy- and light-chain transgenes encoding anti-HEL antibody; in normal mice, the frequency of HEL-reactive B cells is on the order of 1 in 103, but in these transgenic mice the rearranged anti-HEL transgene is expressed by 60%-90% of the mature peripheral B cells. Goodnow mated the two groups of transgenics to produce "double-transgenic" offspring carrying both the HEL and anti-HEL transgenes. Goodnow then asked what effect HEL, which is expressed in the periphery but not in the bone marrow, would have upon the development of B cells expressing the anti-HEL transgene.

The Goodnow double-transgenic system has yielded several interesting findings concerning negative selection of

B cells (Table 11-3). He found that double-transgenic mice expressing high levels of HEL (10-9 M) continued to have mature, peripheral B cells bearing anti-HEL membrane antibody, but these B cells were functionally nonresponsive; that is, they were anergic. The flow-cytometric analysis of B cells from double-transgenic mice showed that, while large numbers of anergic anti-HEL cells were present, they expressed IgM at levels about 20-fold lower than anti-HEL single transgenics (Figure 11-12b). Further study demonstrated that the double transgenics had both surface IgM and IgD, indicating that the anergy was induced in mature rather than immature B cells. When these mice were given an immunizing dose of HEL, few anti-HEL plasma cells were induced and the serum anti-HEL titer was low.

To study what would happen if a class I MHC self-antigen were expressed only in the periphery, Nemazee and Burki modified the transgenic system used in the experiments on

Expression of anti-HEL transgene by mature peripheral B cells in single and double-transgenic mice

TABLE 11-3

Membrane Anti-HEL Anti-HEL

Experimental group HEL level anti-HEL PFC/spleen* serum titer*

Anti-HEL single transgenics None + High High

Anti-HEL/HEL single transgenics (Group 1) 10-9 M + Low Low

* Experimental animals were immunized with hen egg-white lysozyme (HEL). Several days later, hemolytic plaque assays for the number of plasma cells secreting anti-HEL antibody were performed and the serum anti-HEL titers were determined. PFC = plaque-forming cells; see Figure 23-1 for a description of the plaque assay.

SOURCE: Adapted from C. C. Goodnow, 1992, Annu. Rev. Immunol. 10:489.

T3 C

Mice with liver-specific Kb transgene

Mice with anti-Kb transgene

Double transgenics with Kb and anti-Kbtransgene

Single transgenic (Anti-Kö )

Bone marrow

Bone marrow

O

O

T3 C

O

o

mIgM

Lymph node mIgM

Lymph node

O

o

Lymph node

o

negative selection in the bone marrow described previously (Figure 11-5a). They first produced a transgene consisting of the class I K gene linked to a liver-specific promoter, so that the class I K molecule could be expressed only in the liver. Transgenic mice expressing an anti-K antibody on their B cells also were produced, and the two groups of transgenic mice were then mated (Figure 11-13a). In the resulting double-transgenic mice, the immature B cells expressing anti-K mIgM would not encounter class I K molecules in the bone marrow. Flow-cytometric analysis of the B cells in the double transgenics showed that immature B cells expressing the transgene-encoded anti-Kb cells were present in the bone marrow but not in the peripheral lymphoid organs (Figure 11-13b). In the previous experiments of Nemazee and Burki, the class I MHC self-antigen (H-2*) was expressed on all nucleated cells, and immature B cells expressing the transgene-encoded antibody to this class I molecule were selected against and deleted in the bone marrow (see Figure 11-5a). In their second system, however, the class I self-antigen (K) was expressed only in the liver, so that negative selection and deletion occurred at the mature B-cell stage in the periphery.

mIgM

mIgM

FIGURE 11-13

Experimental demonstration of clonal deletion of self-reactive mature peripheral B cells by Nemazee and Burki. (a) Production of double-transgenic mice expressing the class I Kb molecule and anti-Kb antibody. Because the Kb transgene contained a liver-specific promoter, Kb was not expressed in the bone marrow of the transgenics. (b) Flow-cytometric analysis of bone marrow and peripheral (lymph node) B cells for Kb binding versus membrane IgM (mIgM). In the double transgenics, B cells expressing anti-Kb (blue) were present in the bone marrow but were absent in the lymph nodes, indicating that mature self-reactive B cells were deleted in the periphery.

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  • Joe
    How does negative selection affect mature b cells?
    8 years ago

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