LITOLOGÍA

6 . 1 Introduction

Since the results presented in the previous chapter demonstrate that thymocytes bearing H- 2Kb-specific TCRs are deleted in CD2Ki^ x DES and CD2Ki^ x BM3.6 FI mice, it might seem unnecessary to test T cells from CD2Kh mice for their ability to respond to H-2Ki^ in vitro. However, Sponaas et al. (1994b) showed that studies with TCR-tg mice expressing one clonotype could not predict the fate of T cells bearing different clonotypes conferring a particular MHC I specificity. Simpson et al. (1993) carried out cytotoxicity assays on T cells from CD2Kh-3 and CD2Kh-2 mice and detected H-2Kh-reactive T cells from CD2Rh-3 in vitro, but there was no sign of autoreactivity and no response to skin grafts bearing H-2Kh. H-2Kh-reactive T cells were not detected among CD2Kh-2 splenocytes, and since detailed analyses of the pattern of H-2Kh expression was not carried out on these mice, the cause of this difference was attributed to the difference in transgene copy number between the two mouse lines. At the time CD2K^-3 mice were estimated to have four copies, and CD2Kh-2 ten copies (Simpson et al., 1993). However, more accurate measurement of copy number suggests that CD2K^-3 mice only carry two copies (3.2.3; Figure 2). The other CD2K^ lines were generated to determine the number of copies required to reach the threshold after which self-reactive T cells are not detected. This chapter will describe experiments carried out on all available CD2K^ lines.

6 . 2 Assays for Cytotoxicity Mediated by Splenocytes from CD2Kb Mice

and 3.3). Other lines that had siblings with different phenotypes initially (CD2K^-1, CD2K^-7 and CD2K^-11) now resembled each other closely enough for the CTL assay results to be considered together.

The results from these CTL assays are summarised in Table x. The figures shown were obtained from regression analyses carried out by the O M l regression analysis program, underlining indicates values that have an greater than 0.7, when plotted against effector to target ratio. values were average values from the triple experiment carried out on each mouse line, y values measures the extent to which target lysis has taken place. Where possible, experiments where spontaneous lysis of targets makes up 30% or more of the total lysis have been excluded as technical failure. In some cases they have been included in Table x: points indicated with an asterisk (*) have been distorted by spontaneous release of ^^Cr, accounting for 30% or more of total release. A response by splenocytes stimulated with antigen is indicated in table ix by a high y value (ideally >15), with an R^ value greater than 0.7.

Table x shows that, with two notable exceptions, CD2K^ mice do not respond to H-2K^ on CBK splenocytes by producing CTLs (column 2). One of the two exceptions is the CD2K^-3 experiment, included to confirm the results of Simpson et al. (1993), and the other is an experiment with a CD2K^-4.4 (CD2K^-4 line) mouse which, despite testing positive for the transgene by PCR, produced CTLs in response to stimulation in vitro with CBK splenocytes. Possible reasons for this will be discussed below. Two experiments on CD2K^-4.4 mice had already been carried out, neither detected CTL precursors in the CD2K^-4.4 mice. A further two experiments were carried out on CD2K^-4.4 mice, neither of which responded to CBK stimulation by producing CTLs.

Figure 16A shows the ^^Cr release from CBA and a CD2K^-4.4 mouse at effector to target ratios of 30:1,10:1. 3:1 and 1:1. A similar result was obtained from four out of five CD2K^-4.4 mice. Figures 16B and 16C show graphs where effector to target ratio is plotted against mean specific lysis for CD2K^-7 and CD2K^-11 cells stimulated with CBK, compared to release from CBA/Ca effector cells stimulated with CBK.

BALB/c or PIH TR (H-2^) targets were used to monitor any cross-reactive killing by CD2K^ or CBA/Ca CTLs, to ensure that the lysis observed was antigen-specific (Table x). Stimulation of CD2K^ and CBA/Ca splenocytes with BALB/c splenocytes confirmed that the cells from the experimental mice were capable of responding to stimuli and generating CTLs. CD2K^ cells stimulated with BALB/c cells appear to cross-react with H-2^ targets with greater frequency than CD2K^ cells stimulated with CBK on H-2^ targets, though the reasons for this are unknown.

6 . 3 Transgene Copy number Affects the Survival o f Self-reactive T C ells

Comparisons between the levels of H-2K^ expression on PBLs from CD2K^-3 heterozygotes and homozygotes (data not shown) had shown that the increase in transgene copy number led to a corresponding increase in expression (MRFI). CTL assays on splenocytes from CD2K^-3 heterozygotes and homozygotes were carried out, which showed that the number of CTL precursors present among splenocytes from CD2K^-3 homozygotes, were considerably less than the number present in heterozygote spleens. This is illustrated in Figure 17A, which compares the mean specific release from EL4 targets incubated with various numbers of CD2K^-3 heterozygote and homozygote effector cells. At the effector to target ratio 10:1, CBA/Ca splenocytes stimulated with irradiated CBK splenocytes, gave 25.6% mean specific lysis, with an value of >0.7. CD2K^-3 heterozygotes had 13.5% lysis at effector to target ratio 10:1, with an R^ value >0.7. In contrast, CD2K^-3 homozygote mice possessed splenocytes that lysed merely 6.4% at 10:1 effector to target ratio, with an R^ value less than 0.7. Thus, splenocytes from CD2K^-3 heterozygote mice include cells capable of lysing labelled EL4 targets, while less self-reactive cells are present in the periphery of CD2K^-3 homozygote mice.

This data was confirmed by ELISA assays for IFNy release from lymph node cells from CBA/Ca, CD2K^-3 heterozygote and homozygote mice, stimulated with CBK

responding to H-2K^, but cells from CD2K^-3 homozygotes released less IFNy than those from heterozygote or CBA/Ca mice.

Table x Suir Responders

imary of CTL Assays carried out stimulated with CBK cells H-2^ targets H-2^

on Splenocy targets

es from CD2K1^ Mice

stimulated with BALB/c cells H-2b targets H-2^ targets CD2K»-1 1.9 (29.3)1 16.7 5.1 23.2* (19.1) CD2Kb-l 9.4 (2 0.1) 5.8 (11.4) NT NT CD2Kb-3 15,9 142,5.) 1L2 (37.6)2 19,7 (12.0) 16.8 (19.7) C D 2K M 2 2.7 (19.8) 2.7 3.6 (Z3) 52.0* (35.8) CD2Kb-42 4.7 (16.7) 3.6 (3.7) NT NT CD2Kb-42 8.5 (32.5) 5.4 (10.9) NT NT CD2Kb-44 3.4 (24,0) 3.1 (16.6) 30.9 (16.0) 42.7* (23.2) CD2Kb-44 28.6 (32,5) 13.2 (10.9) NT NT CD2Kb-44 9.6 (41,3) NT NT NT CD2Kb-44 6.4 (41.3) NT NT NT CD2K*>-5 9.1 (42.1) NT NT 21.3* (35.3)* CD2Kb-7 12.1 (35.4) NT 21.5 NT (1 0.1) CD2Kb-7 3.9 (19.9) 14.5 (7.5) NT NT CD 2K b-ll 5.8 (20,5) 9.4 24.3* 28.8 (27.1) CD 2K b-ll 4.9 (9.9) 7.1 5.3 32.2 (33.9) CT)2Kb-ll 8 .8 (12.3) 8.1 (2 2.6) NT NT CD 2K b-ll 8 .2 (28.1) 6.1 _(19-J) NT NT

F ig u re 16

E x a m p les o f C T L A ssays c a rrie d o u t on Splenocytes fro m CD2K*^-4, C D 2 K b -7 a n d C D 2 K b -Il Mice

This figure shows three of the CTL assays carried out on CD2K^ mice: A, CD2K^-4, B, CD2K^-7 and C, CD2K^-11. In each case the cytotoxic activity of T cells from the CD2K^-3 mouse (squares) against EL4 cells after stimulation with H-2Kb on CBK cells is compared to cytotoxic activity of CBA/Ca cells (circles) after stimulation with H-2K^. CTL assays were carried out as described (2.9.3), and these graphs were obtained by regression analyses performed using the OMl program.

B

Figure 17

T ran sg en e C opy N u m b er D irectly Affects Survival of Self-Reactive T Cells

This figure shows the results of two different assays that compare the ability of CD2K^-3 heterozygotes and homozygotes to respond to H-2Kt>. Graph A compares the cytotoxic activity of CBA/Ca cells ( filled circles )) and cells from CD2K^-3 heterozygotes (square^ and homozygotes (open squares) against EL4 cells after stimulation with H-2K^> on CBK cells. Graph B compares the IFNy release by cells ft’om CD2K^-3 heterozygotes (circles) and homozygotes (open squares) to that of CBA/Ca (filled squares) cells stimulated with H -2K ^ on CBK cells. On graph B each point is the mean of three CD readings, and serially diluted supernatant was added to the ELISA.

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