Thyrocytes were initially cultured according to two different protocols to determine which was most suitable to assess presentation to Tg specific hybridomas. The first method was that used by Yeni & Charreire (1981), where thyrocytes were used as antigen presenting cells from day five of culture and cultured in a simplified basal medium. The method previously used by workers in the Department of Immunology used thyrocytes from day 12 of culture and a complicated, hormone supplemented growth medium. These two methods were compared using the basal medium described by Yeni & Charreire (1981). Figure 5.1 A shows the levels of presentation achieved using these two time points. Panel
a) shows levels of presentation to CH9 by CBA/Ca thyroid epithelial cells cultured for five days before use. It can be seen that in these experiments mouse thyroglobulin is not processed and presented by the TEC’s in the presence or absence of IFN-y. This is in contrast to the 11 mer T4+(2553) peptide which is presented by the TEC’s in the absence of IFN-y, this may indicate the presence of a small amount of class II already present on the cell surface. However this effect is substantially up-regulated by the addition of 100 U/ml IFN-y to the culture medium for 72 h prior to assay.
Thyroid epithelial cells are able to synthesise Tg in culture and therefore might have been able to present endogenously derived peptide by the class II pathway. Given the requirement for iodinated Tg for the induction and recognition of the pathogenic epitope, sodium iodide (Nal) at a final concentration of IjiM was incorporated into the culture medium to ensure that if epitopes were processed they would be fully iodinated and therefore be recognised. In this experiment there appears to be a small degree of endogenous presentation in the presence of Nal which is statistically significant. In other experiments no endogenous presentation was seen, thus this phenomenon seems to be unreproducible. Although the level of endogenous presentation was statistically significant it is probably not biologically
significant. Figure 5. IB panel a) shows an example of a number of experiments which gave the same result, however there were other experiments that exhibited the same pattern of results as in 6) therefore the results at 12 days did not appear to be very consistent. This may have been due to the long culture time allowing the outgrowth of contaminating cells such as fibroblasts. No quantification was made as to the com parative levels of contam ination between five and twelve day culture, although by viewing, it could be seen that there were indeed more contaminating fibroblastic cells after twelve days than at five.
a). IL-2 release (CTLL cpm xlO"^)
1000 A n tigen MTg 50ug/m! T4+(2553) 5pg/ml *** *** Medium
b). IL-2 release (CTLL cpm xlO"^)
A n tigen IF N y MTg 50pg/ml T4+C2553) 5pg/ml Nal IpM Medium + + Day 12
Figure: - 5.1 A. The effect of culture period.
Response of 2x10^ CH9 to antigenic presentation by CBA/Ca thyroid epithelial cell cultures in the presence or absence of IFN-y as indicated in the figure. Responses to 50pg MTg ( - g - ) , 5 p.g T4+(2553) peptide ( -g -), IpM Nal (-gj-) and medium control ( - g - ) are shown.
(a) shows the effect of culture of TEC for 5 days prior to assay and (b) shows the effect of culture of TEC for 12 days prior to assay. Supernatants taken after 24h , for assessment of IL-2 release, results shown are average of triplicate cultures +/- SEM. (Non significant values not indicated * p<0.05, ** p<0.01 and ***p<0.001). See Appendix 1.
5. IB) The effect of culture medium.
The two different growth mediums (basal and hormonal supplemented) were com pared using the five day culture period. Figure 3.3B shows that the growth medium had no effect on the levels of presentation by the T E C ’s of the T4+(2553) peptide in the presence or absence of IFN-y. Both culture mediums maintain the differential in presentation seen on the ad d itio n of IFN -y and th ere is no p resen ta tio n o f ex o g en o u s or en d o g en o u s thyroglobulin. Therefore it was decided to use the simplified growth medium and the five day culture period as employed by Yeni & Charreire (1981) which will be referred to TEC
RPMI 5%.
IL-2 release (CTLL cpmxlO"^)
5 10 15 2 0 J I L A n tigen T4+(2553) 5g g /m l T4+(2553) 5g g /m l M edium M edium
Figure:- 5.IB. The effect of culture medium.
Response of 2x10^ CH9 to antigenic presentation by 5 day CBA /Ca thyroid epithelial cell cultures in the presence or absence of IFN-y as indicated in the figure. Responses to 50|ig MTg, 5 pg T4+(2553) peptide, IpM N al and medium control are as indicated in the graph. The effect of culture medium TEC 5% ( - g - ) and TEC 5% supplem ented with hormones (-gg-) as indicated in section 2.2.3 are shown. Supernatants taken after 24h, for assessment of IL-2 release, results shown are average of triplicate cultures +/- SEM. See Appendix 2.
5.2 The presence of ATA does not improve thyroid récognition.
It was postulated that any increases in endogenous presentationmaay have been occurring was masked by high control levels of presentation as a result of iodinated thyroglobulin being present in the cells at the time of culture. If this could be removed prior to culture any newly synthesised Tg and subsequent presentation of endogenous peptide would be seen, particularly if Nal was added to the culture medium to allow any endogenously produced Tg to be fully iodinated. Initially thyroids were taken from ATA treated mice which although still making Tg, would be iodine depleted. However it proved impossible to prepare thyroid follicles from these thyroids due to the extensive hyperplasia in the glands. Even on minimal digestion, glands were totally digested resulting in a single cell
suspension containing a small number of follicles. These follicles on plating did not adhere as did normal follicles and therefore did not spread to form a monolayer. Therefore it was decided to take normal mouse thyroid glands and treat them from day two after setting up of the cultures with a 1 % solution of ATA in the culture medium with the view that newly synthesised Tg may be iodine deficient unless l|iM N al was added to the culture medium at the same time.
Figure 5.2 shows the effect of adding ATA to the culture medium. In panel a), the presence of ATA does not facilitate the detection o f peptide processed from exogenously or endogenously derived peptide and levels of proliferation are not significantly elevated above background. However the presence of ATA appears to elevate the levels o f T4+(2553) peptide that are presented in the presence of IFN-y. This is in contrast to other experimental results that show that the presence of ATA has no effect on the levels o f T4+(2553) peptide presentation in the presence of IFN-y as shown in panel b). There is no significant increase in the levels of presentation of endogenous or exogenous Tg. Thus the presence of ATA appears not to have any obvious beneficial effect and was therefore not used in routine thvroid cell culture.
IL-2 release (CTLL cpm xlO"-^)
IFN-y ATA Antigen M Tg 50 |ig/m l TZZZZZZZZZZA-^ Nal |iM Medium
Figure:- 5.2. The presence of ATA does not consistently improve thvroid antigen recognition.
Response of 2x10^ CH9 to antigenic presentation by 5 day CBA /Ca thyroid epithelial cell cultures in the presence or absence of 1% ATA in the culture m edium from day 1, as indicated in the figure, the presence or absence of IFN-y is also indicated. Responses to 50pg MTg ( - g - ) , 5 pg T4+(2553) peptide ( - ^ - ) , IpM N al (-[!]-) and medium control ( - g - ) are shown. Panels a) and h) (shown on the next page) show representative repeat experiments and illustrate the effect of ATA on the presentation assay. Supernatants taken after 24h, for assessment of IL-2 release, results shown are average of triplicate cultures +/- SEM. Figure 5.2b is shown on the next page. (Non-significant values not indicated *p<0.05, **p<0.01 and ***p<0.001). See Appendix 3
b). A n tigen M T g 5 0 |ig /m l T 4 + (2 5 5 3 ) 5 |J.g/ml Nal 1 |iM M edium I F N -y + + 4- 4- 4- 4- 4- 4- A T A
IL-2 release (CTLL cpm xlO"^)
10 20 30 40 50
5.3 The presence of TSH does not improve thyroid antigen recognition.
The absence of presentation of exogenously derived Tg may have been due to reduced endocytosis from the tissue culture medium; therefore following monolayer preparation the cells were incubated from day two in culture medium that had been supplemented by 0.1 pg/ml TSH, with a view to stimulating uptake of Tg from the surrounding medium. Figure 5.3 shows CBA/Ca derived thyrocytes presenting to CH9 in the presence or absence of TSH. It can be seen that TSH does not have any significant effect on the processing and subsequent presentation of Tg derived peptide, normal spleen cell controls for this experiment show recognition of Tg by CH9. There is no increase in the presentation of T 4+ (2553) peptide to CH 9, but there may be a sm all, but sign ificant, increase in endogenously derived antigen presentation in the presence of TSH. However this level of stim ulation is probably not biologically significant. The presentation of exogenously derived MTg was also not improved by the addition of up to 500pg/m l MTg prior to assay of presentation to CH9; TSH was also unable to improve this presentation (data not shown). These levels in culture are high enough to be comparable to that which would be obtained
i n t h e t h y r o i d f o l l i c l e i n v / v o . A n tig e n MTg 50 |ig/ml T 4+(2553) 5 u g /m i N a l 1 ^iM M edium TSH IF N -y I L -2 r e le a s e (C T L L c p m x lO '^ ) 5 10 15 20
Figure:- 5.3. The presence of TSH does not improve thvroid antigen recognition. Response of 2x10^ CH9 to antigenic presentation by 5 day CBA/Ca thyroid epithelial cell cultures in the presence or absence of 0. lp.g/ml TSH in the culture m edium from day 1, as indicated in the figure, the presence or absence of IFN-y is also indicated. Responses to 50|ig MTg (-Q -), 5 p,g T4+(2553) peptide ( - ^ - ) , IjiM Nal (-[J-) and medium control ( - g - ) are shown. Supernatants taken after 24hi, for assessment o f IL-2 release, results shown are average of triplicate cultures +/- SEM. (Non significant values not indicated * p<0.05, ** p<0.01 and ***p<0.001). See Appendix 4.