Diferencias entre modelo completo no lineal y modelo

I - b . 7 . 6 - b . 7 . 6 + IL -4 I - A k 7 I - A k 7 + IL -4 1 - 1 . 1 9 I - 1 . 1 9 + IL -4 I - J A 1 2 . 5 J A 1 2 . 5 + I L -4 - I —

20

100

Figure 4.19. Effects of co-stimulation with anti-CD40 and/or IL-4 on the induction of apoptosis in B cells. B cells (at lO^/ml) were cultured for 16 h with medium alone, with 10 ^g/ml bio-anti-ô + 10 /xg/ml avidin, in the presence or absence of anti-CD40 mAb (10 /xg/ml) and/or IL-4 (10 U/ml). They were then stained with PI, and nuclei with subdiploid levels of DNA were assayed by flow cytometry as described in section 2.4.2.

% A p o p to tic nuclei 0 10 2 0 3 0 4 0 5 0 6 0 7 0 Medium b i o - a n t i - 6 / A v I L - 4 I L - 4 / b i o - a n t i - 6 / A v a n t i - C D 4 0 Ant i - CD 4 0 / b i o - a n t i - c 5 / A v I L - 4 / C D 4 0 I L - 4 / C D 4 0 / b i o - a n t i - 6 / A v

Table 4.1

TIME-COURSE OF THE APPEARANCE OF APOPTOTIC B CELLS AFTER CULTURE WITH CROSSLINKED ANTI-Ig ANTIBODIES

Treatment*^ Percentage of apoptotic nuclei at?*^

4 hr Shr 16 hr 24 hr Medium 18 23 12 12 Avidin 17 26 14 9 Anti-f4 12 23 17 22 Anti-/z/Av 32 49 43 37 Anti-ô 14 23 20 28 Anti-ô/Av 47 52 41 61 Anti-Cl. I 17 24 10 12 Anti-Cl. I/Av 19 30 12 16

a) B cells were cultured for the indicated periods of time at 10^/ml in medium, or with 10 fig/ml of the indicated biotinylated mAh in the presence or absence of 20 /ig/ml avidin. The cultures were then stained with PI as described in Section 2.4.3.

b) Percentages of cells showing less than 2C DNA, as illustrated in Fig. 4.13.

Table 4.2

EFFECTS OF CROSSLINKING sIg WITH IMMOBILISED ANTIBODY FOR 24 H

Marker Control*^ i-anti-ô i-dnti-fi

% positive MFI % positive MFI % positive MFI

sIgM<^> 99 681 99 673 72 579

sIgD 93 905 92 857 77 716

Class I 99 837 99 821 85 848

Class II 99 585 99 664 77 701

CD45 99 909 99 921 86 876

a) B cells (10^/ml) were cultured for 24 h in medium, or with i-anti-Ig (50^g/ml). They were then washed and stained with fluroescein-labelled mAh to IgM (Ak4), IgD (1.19) CD45 (M l/9.3). Class II (NIMR-4) or Class I (11.4.1). Cells were analysed in a FACScan Flow cytometer, gating on viable cells by setting appropriate scatter gates.

b) Data are presented as percentages of stained cells and median fluorescence intensities (MFI).

Table 4.3

EFFECTS OF HYPERCROSSLINKING sIg RECEPTORS WITH BIO-ANTI-Ig PLUS AVIDIN

Marker Medium*) % positive MFI Avidin % positive MFI Bio-anti % positive MFI Bio-anti-/x/Av % positive MFI Bio-anti-ô % positive MFI Bio-anti-ô/Av % positive MFI IgM*') 95 789 96 785 95 770 96 777 94 772 94 709 IgD 96 802 95 789 95 699 96 754 95 549 97 755 Class I 96 739 96 720 97 731 97 759 95 718 96 736 Class II 95 706 94 690 95 815 96 815 95 808 95 749 CD45 93 729 95 738 95 715 94 702 94 693 93 641

a) B cells (10^/ml) were cultured for 16 h in medium, or bio-anti-Ig (10 /xg/ml), in the presence or absence of avidin (20 /xg/ml); they were washed and stained with fluorescein-labelled mAb to IgM (AK4), IgD (1.19), CD45 (Ml/9.3), Class II (NIMR-4) or Class I (11.4.1). Cells were analysed in a FACScan flow cytometer, gating on viable cells by setting appropriate scatter gates.

b) Data are presented as percentages of stained cells and median fluorescence intensities (MFI).

4.13.1. Discussion part I: The role of sIgM and sIgD in mature B cell tolerance.

As discussed in section 1.6, hypermutation of Ig genes in mature peripheral B cells can potentially result in the generation of high affinity autoreactive cells, thus necessitating a mechanism for tolerance that can silence B cells at this stage of their differentiation. It is clear that sIg receptors on mature B cells have the capacity to generate transmembrane signals that either promote or inhibit cellular growth and differentiation. For example, crosslinking of sIgM or sIgD on mature B cells normally induces DNA synthesis and B cell activation (reviewed in Cambier and Ransom, 1987, Klaus et a l , 1987). However, sIg crosslinking can also result in the inhibition of polyclonal antibody production in co-culture experiments with antigen or mitogen (Flahart and Lawton, 1987, Warner and Scott, 1991), and recently transgenic mouse models have demonstrated Ig-mediated negative signalling in the induction of mature B cell tolerance (Russell et al. , 1989, Goodnow et al. , 1989). We have developed two models to investigate mature B cell unresponsiveness, and the results presented here show that resting splenic B cells can be rendered unresponsive to subsequent mitogenic stimuli by hypercrosslinking of either class of sIg receptor.

Prolonged exposure to immobilised anti-Ig Ab inhibited the responses to heterologous anti-Ig mAb or LPS (Figs. 4.1 and 4.3), inducing polyclonal unresponsiveness and eventually resulting in apoptotic cell death. Although the immobilised Ab system may provide a model for the response to membrane bound antigen, it probably does not reflect the effects of multivalent soluble antigens. To this end, a second system of extensively crosslinking cell surface molecules was tested, using soluble, biotinylated anti-Ig Abs crosslinked on the cell surface with avidin (or in some experiments streptavidin). The results with this model indicate that

normally mitogenic anti-Ig mAb when hypercrosslinked using the biotin/avidin system similarly induce dose dependent polyclonal unresponsiveness and cell death in mature B cells (Figs. 4.4 and 4.5).

The kinetics of induction of unresponsiveness to heterologous anti-Ig mAb were considerably slower in the immobilised Ab system (Figs. 4.3 and 4.5), which suggests the bio-Ab plus avidin system induces a more potent inhibitory signal. Anderssen et al. (1977) have shown that only approximately 30% of splenic B cells are responsive to LPS. Results obtained here indicate that 48 h preculture with hypercrosslinked Abs (in both systems) was necessary to induce unresponsiveness to LPS in this subpopulation. The biochemical signalling pathway triggered by LPS is not yet known, but these data indicate that it is less sensitive to the hypercrosslinked Ab-mediated inhibitory signal than the Ig receptor-mediated activation signal.

Ligation of sIgM or sIgD receptors induced seemingly identical effects over a 72 h culture period in both models (Figs. 4.1 and 4.5), in agreement with a recent paper by Brink et at., (1992). However, kinetic studies indicated that crosslinking sIgD produced inhibitory effects more rapidly. A 4 h pulse with crosslinked bio-1.19 reproducibly caused 50 - 90% inhibition, whereas a corresponding treatment with anti-^ was ineffective (Fig. 4.7). It is not clear whether this effect was due to IgD mediating a stronger signal, or simply a reflection of the fact that most mature B cells express more sIgD than sIgM (Vitetta and Uhr, 1975). A similar observation of the apparently stronger IgD-mediated signal occurred when anti-^ or anti-ô Abs were crosslinked with streptavidin. All but the highest ratio of anti-/i : streptavidin caused activation, whereas streptavidin-crosslinked anti-ô consistently induced inhibition of DNA synthesis (Fig. 4.9). This contrasts to a study by Gaur et al. (1993), demonstrating that anti-/x, but not anti-ô, inhibited the LPS driven differentiation of

mature B cells, although anti-ô synergised with anti-f* to induce unresponsiveness. However, Gaur et al. used soluble anti-Ig Abs, and it is possible that inadequate crosslinking of Ig receptors accounts for the discrepancy in results. Additionally, as there are marked differences between the two experimental systems, different inhibitory mechanisms may be involved.

The observed abrogation of DNA synthesis caused by hypercrosslinked anti-Ig Ab might be explained in two ways. One possibility was that hypercrosslinking of sIg receptors resulted in apoptosis leading to clonal deletion (see section 1.6). Alternatively, as it is known that conventional crosslinking of one isotype of sIg causes temporary desensitization of the signalling pathway associated with the heterologous Ig receptor (Cambier et a l , 1988, Klaus et al.y 1989), it was possible that extensive crosslinking of sIg caused a more long term heterologous desensitization. This might be related to the long-lasting anergy observed in transgenic mouse models induced by oligovalent antigen (Goodnow et al. , 1988 and 1989). However, two different detection methods showed that hypercrosslinking sIg on mature B cells induced apoptotic death, which was detectable within 4 h (Table 4.1 and Fig. 4.15), and eventually virtually all the cells undergo apoptosis (Figs. 4.13 and 4.14). Experiments investigating reversibility of the negative effect indicated that B cells did not recover their normal proliferative rate if the hypercrosslinked anti-Ig was removed (Fig. 4.17), and suggest that cells become committed to the tolerance pathway. However, subsequent to completion of this report, further experiments in the laboratory using the same protocol show that i-anti fj, treated cells can proliferate in response to s-anti-0 following removal of the inhibitory reagent. The proliferative response peaked much earlier than in control cells, and decreased dramatically by 48 h after washing out the crosslinked antibody (M Holman, unpublished data). This

would account for the inability to detect a response at 72 h, as described in this chapter. Together these results suggest that the i-anti-Ig primes mature B cells to respond more rapidly, in addition to triggering the apoptotic pathway.

Goodnow et al. (1988 and 1989) have shown that anergic B lymphocytes express high levels of sIgD but a 10 - 20 fold reduction of sIgM. Mature B cells cultured for 16 - 24 h with hypercrosslinked anti-Ig Ab in either system did not show any marked reduction in sIgM (or sIgD) expression, although as it appears that the cells are undergoing apoptosis rather than anergy this was not unexpected. Interestingly, the cells which survive for 12 - 24 h appear to have been abortively activated, as evidenced by their increased levels of class II antigen (Tables 4.2 and 4.3). However, pHJUrd and pH]TdR uptake assays showed that both RNA and DNA synthesis were progressively reduced during the unresponsive state (Figs. 4.2 and 4.4), suggesting that the cells do not enter the Gi phase of the cell cycle.

An important conclusion from this study is that the degree of crosslinking of sIg receptors in mature B cells determines whether the cells are activated or deleted. These results are in line with the signalling threshold model of B cell activation and tolerance briefly discussed in section 1.6. This model was largely based on data using TI antigens, which greatly simplified the experimental situation by inducing B cell stimulation directly, without the need for Th cells. Later work, particularly the development of transgenic mouse models, has also shown its application to TD antigens. The model proposes that the B cell response is determined mainly by the epitope density (or sIg crosslinking capacity) of a given antigen (reviewed in Klaus et al., 1976, Weigel and Perelson, 1981, Goodnow, 1992). An antigen with epitope density below a critical level will result in insufficient sIg crosslinking to induce a response, and Goodnow et at. (1989), using the HEL transgenic model, suggest that

when <5% of antigen receptors are ligated by HEL such clonal ignorance results. Above this critical threshold of epitope density, antigens become immunogens, inducing sufficient crosslinking to induce B cell activation (for example Dintzis et al. , 1976). This level of crosslinking is described as "restrictive" in the Weigel and Perelson model (1981). At very high epitope density antigens are thought to become obligate tolerogens, crosslinking a high percentage of antigen receptors, locking the receptor into position and preventing any lateral movement in the membrane (Peacock and Barisas, 1981). This extensive crosslinking of sIg is sometimes described as overly restrictive (Weigel and Perelson, 1981). Distinct signalling thresholds for the induction of clonal anergy and clonal deletion are believed to occur within the overly restrictive type of sig-hypercrosslinking. Multivalent antigens with high epitope density, and recognised with high affinity induce clonal deletion, whilst more poorly crosslinking antigens, recognised with relatively low affinity induce anergy (Goodnow et a l , 1989, Nemazee et al., 1989a and b. Hartley et a l , 1991, reviewed in Goodnow, 1992).

In agreement with the receptor signalling threshold model, the results suggest a distinct threshold of sIg crosslinking, above which tolerance is induced. Below this threshold DNA synthesis and cell proliferation appear to result, as it was noted in titration experiments that the mitogenic responses to suboptimal concentrations ofbio- antx-fi were enhanced by crosslinking with a critical concentration of avidin (data not shown) or streptavidin (Fig. 4.9). A greater degree of crosslinking, caused by increasing the concentration of anti-Ig and/or avidin or streptavidin, resulted in inhibition. The immobilised anti-Ig system described here can easily be envisaged as inducing overly restrictive crosslinking, with subsequent immobilisation of antigen receptors under conditions where the B cell is unable to clear such an antigen lattice

from the membrane (by shedding or endocytosis), as it would normally do during the events leading up to triggering. The bio-anti-Ig plus avidin system also appears to induce the overly restrictive crosslinldng necessary for negative signalling, but it is not yet clear whether the sig-bio-anti-lg plus avidin complexes are subsequently shed or endocytosed. It would be interesting to investigate this point in future experiments.

The degree of multimerization of sIg receptors is influenced by a number of factors in addition to epitope density/capacity to crosslink sIg, in particular antibody affinity and antigen concentration (Mongini et al, y 1991 and 1992, reviewed in Goodnow, 1992). Results presented here show that higher concentrations of uncrosslinked bio-anti-/x or anti-ô Abs caused some increases in the background level of apoptosis, suggesting that soluble Abs may also cause the critical level of sIg crosslinldng on some B cells to induce apoptosis if present in sufficient quantity. This further indicates the importance of antigen concentration in B cell tolerance induction. The signalling threshold model as described above, does not explain the influence of factors such as antigen concentration and antibody affinity in determining the outcome of antigen binding to sIg. Much more work is necessary to investigate precisely how these physiochemical properties of antigen and sIg receptors combine to induce activation, anergy or tolerance in B cells.

B cell activation from a resting Gq state is characterised by several sequential stages, the first stage a transitional excitation state between Go and G^ termed G ^ (Klaus et al. y 1985), and characterised by increased class II antigen expression and cell enlargement. With continued crosslinldng of sIg, some transitionally activated B cells are induced to enter Gi, where the overall rate of mRNA synthesis is increased, and cytokines and cytokine receptors are expressed. Even in the absence of T cell derived factors, a proportion of B cells activated by Ig mediated signalling can

progress into S phase (Goroff et a l , 1986, Mongini et a l , 1991 and 1992). Data presented here demonstrate that hypercrosslinking of sIg receptors induces cells to pass into the Git state, as shown by their increased MHC Class II expression, but does not result in RNA or DNA synthesis, suggesting they subsequently follow an apoptotic pathway. Interestingly, the non-mitogenic Abs AK4, AK7 and JA12.5 caused identical inhibitory effects to the mitogenic Abs (Fig. 4.1). Leptin (1985) has shown that AK4 and AK7 induce B cell activation when coupled to Sepharose, and the present results show that hypercrosslinking these so called non-mitogenic antibodies induced massive growth inhibition and apoptosis. Such results also reinforce the signalling threshold model, with moderate crosslinldng of antigens, influenced by antibody affinity and antigen loading, causing oligomerization of sIg receptors, and resulting in cell proliferation. Hypercrosslinking of antigen receptors by multivalent antigen at high concentration and/or recognised with high affinity, induces receptor multimerization, and results in apoptotic deletion. Perhaps the critical threshold for inducing anergy, as described by Goodnow et a l , 1988 and

1989, falls between these two levels of receptor clustering.

The observations reported here were unexpected as previous reports had indicated that extensive crosslinldng of sIg receptors favours B cell activation (Parker, 1975, Pure and Vitetta, 1980, Leptin, 1985, Brunswick et a/., 1988). However, although some previous models may appear superficially similar to the biotin/avidin or immobilised Ab systems, there are differences which could explain the differences observed experimentally. One model which has frequently been used to activate B cells is anti-Ig Ab bound to Sepharose or polyacrylamide beads (Parker, 1975, Pure and Vitetta, 1980, Leptin, 1985). In this system, the size of the beads would preclude the engagement of more than a fraction of total sIg receptors on each cell, hence