TABLA Nº 2: CONSTITUYENTES ORGANICOS RAPIDAMENTE Y

Supernatants taken from the cultures prior to the addition of thymidine were also assayed for IL4 by determining their ability to induce class II expression on splenic B cells. Class II induction was measured after 24-48 hours by determining the mean fluorescence intensity of staining with an anti class II antibody. In general, higher levels of IL4 were detectable in those cultures stimulated with anti CD3 than with PHA but this depended on the absolute level of proliferation. Figure 6.5 shows the titration of supernatants obtained from cells cultured for 3 days in the presence of PHA and feeders. Results are expressed as mean fluorescence intensity. In this experiment, only enough CD45RA+ cells were retrieved to be able to set up a bulk culture whereas the CD4+ and CD45RA- cells were cultured in 96 well plates as well. As can be seen from the titration, at this stage of the culture only the CD4+ and CD45RA- subsets stimulated with PHA and feeders produced IL4 above background levels. However, it cannot be assessed how well the CD45RA+ subset was proliferating. It is worth noting that the unseparated CD4+ subset produces a higher level of IL4 than the CD45RA- subset. This might suggest that the CD45RA+ subset augments production of IL4 by the CD45RA- subset since in this experiment it appeared unable to produce IL4 by itself. However, in other experiments, stimulation with PHA did result in production of IL4 by both CD45RA+ and CD45RA- subsets (e.g. figure 6.6b).

The level of IL4 found in the cultures depended on the degree of proliferation and the nature of the stimulus. In response to anti CD3, both the CD4+CD45RA+ and the CD4+CD45RA- subsets produced IL4 (figure 6.6a). However, the CD4+CD45RA- subset produced more IL4 as judged by the failure to show a fall in fluorescence even at a 1 in 54

Figure 6.5

Titration of supernatants for IL4 activity Day 3 - stimulated with PH A and feeders

1000

-,

CD45RA+ with PHA Feeders with PHA CD45RA-

CD4+

dilution (logS)

Supernatants were removed from cultures and incubated, in serial dilutions, with purified B cells for 48 hours. The B cells were then stained with FITC labelled anti Class II mAb and the results are expressed as the mean fluorescence intensity of class II staining. Controls included supernatants from purified subsets without added PHA, irradiated feeders and PHA without added purified cells. Each plate of cultured B cells was done in duplicate, one with added supernatant and one with supernatant and the anti IL4 mAb, 11B11 to determine the specificity of induction of class II.

Figure 6.6

IL4 titration from CD4+CD45RA+ & CD45RA- cultures stimulated for 4 days with;

a) antiCDS +/- feeders b) PHA +/- feeders

G ) CD

CD45RA+ with feeders

400 n

400 -, _{CD45RA- with feeders}

CD45RA+ CD45RA- 300 - 300 - E 200 - 100 - 100 - 1 5 0 1 2 3 4 5 2 4 0 1 3

dilution (log3) dilution (log3)

T he supernatants from the cultures w ere incubated, in serial dilution, w ith purified B cells fo r 48 hours follow ing w hich the B cells w e re stained with FITC conjugated anti cla ss II m A b to de term in e the level o f induction of IL4. S pecificity of the effect w as de term in ed by parallel cu ltu res in the pre sen ce o f th e anti IL4 an tibo dy, 11B11, w h ich c o m p le te ly abrog ated cla ss II induction. T h e results are e xp resse d as th e m ean level o f fluorescence of th e class II positive cells.

dilution. The induction of class II was due solely to IL4 as judged by complete inhibition by the addition of the anti IL4 mAb, 11B11 (data not shown). Interestingly, in this experiment, the addition of feeder cells augmented the level of IL4 produced by both subsets in response to anti CD3. However, in response to PHA, the CD45RA+ subset produced more IL4 than the CD45RA- subset whether or not feeders were present. This was despite a higher level of proliferation by the CD45RA' subset in the presence of feeders (CD45RA- 44,000cpm, CD45RA+ 19000cpm).

Although the data for IL4 production are somewhat variable in timing and quantity from experiment to experiment, it is clear that both subsets can produce IL4 but that the major producer is the CD4+CD45RA- subset.

Together these data suggest that the CD4+CD45RA+subset is hyporesponsive compared to the CD4+CD45RA- subset and that furthermore, this split does not define the phenotype of Th1 versus Th2 cells. The importance of costimulatory signals in augmenting responses is highlighted by the effect of adding feeders.

6,3 Summ ary of findings in Chapter 6

1. On activation with mitogen or anti CD3 antibodies, the CD4+CD45RA+subset proliferates less well than the CD4+CD45RA- subset when assessed early after activation. However, when stimulated with anti CD3, with time this difference is less marked suggesting a delay in activation rather than an absolute deficiency.

2. Both the CD4+CD45RA+ and CD4+CD45RA' can produce IL2. The CD4+CD45RA' produces IL4 more readily than the CD4+CD45RA+ subset, but the latter is certainly able to produce IL4. Hence, the phenotypic split into CD45RA+ and CD45RA' does not define the Th1/Th2 lymphokine split.