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Syngeneic sensitization of mouse lymphocytes on monolayers of thyroid epithelial cells
CELLULAR
IMMUNOLOGY
78, 387-39 I (1983)
Syngeneic Sensitization of Mouse Lymphocytes Monolayers of Thyroid Epithelial Cells IV. Correlation
on
with H-2 Haplotypes
JEANSALAMEROANDJEANNINECHARREIRE Inserm U 25, Hspital Necker, 161 rue de S&es,
75730 Paris C&dex 15, France
Received December 21, 1982; accepted March 9, 1983 In vitro primary syngeneicsensitization on monolayer of thyroid epithelial cells was performed with 21 inbred strains of mice representing 11 original H-2 haplotypes. Significant differences in the proliferative responses,assessedby thymidine uptake, were found to be related to the major h&compatibility complex haplotype. This result was further confirmed using congenic resistant strains of mice. In comparison with the experimental autoimmune thyroiditis induced by syngeneic thyroglobulin and adjuvant, primary syngeneic sensitization on monolayers of thyroid epithelial cells appeared to be under the same genetic control (H-2k strains being good responders, while H-2b mice are poor responders).
INTRODUCTION During “in vitro” primary syngeneic sensitization (PSS)’ on monolayers of thyroid epithelial cells (1, 2), we demonstrated that T lymphocytes were stimulated (3) and able to induce thyroiditis when injected into normal syngeneic recipients (4). The evidence for immunological sensitization is provided by both the specificity of the stimulation directed only against syngeneic thyroid epithelial cells (TEC) and the production of memory cells detected by a characteristic secondary stimulation on syngeneic monolayers of TEC (2). Using this simple and objective model, we investigated if this stimulation was under genetic control as described for murine experimental autoimmune thyroiditis (5) in which the animals were immunized with thyroglobulin (Tg) and complete Freund’s adjuvant or spontaneous diseasesin rats (6) chickens (7), and humans (8). MATERIALS
AND METHODS
I. Animals. The mice strains AKR (H-2k), C3H (H-2’), CBA (H-2k), A (H-29, SJL (H-2’) DBA/2 (H-2d), BALB/c(H-2d), BALB/b (H-2b), and C57BL/6 (H-2b) were purchased from the Centre de Selection et d’Elevage des Animaux de Laboratoire, CNRS, Orleans la Source, France. The mice strains of the BlO background, BlO.BR ’ Abbreviations used: EAT, experimental autoimmune thyroiditis; FCS, fetal calf serum; HBSS, Hanks’ balanced salt solution; MHC, major histocompatibility complex; NMS, normal mouse serum; ESS,primary syngeneic sensitization; TEC, thyroid epithelial cells; Tg, thyroglobulin. 387 0008-8749/83 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.
388
SHORT
COMMUNICATIONS
(I-I-2k), BIO.A (H-2”), BIOS (H-2S), BlO.D2 (H-2d), BIO.M (H-21, BlO.WB (H-2’), Bl0.P (H-2P), BIO.G (H-2’), BIO.R III (H-23, BlO.PL (H-2U), BlOSM (H-2’), and B 10 (H-2b), were donated by ProfessorColombani, Hopital Saint Louis, Paris, France. All these animals were used between 8 and 16 weeks of age. 2. Lymphoid cell suspensions. Spleen cells were aseptically removed and homogenized in a glasstube with a loose-fitting Teflon pestle. Cell suspensionswere washed twice in Hanks’ balanced salt solution (HBSS) supplemented with penicillin (100 U/ ml), streptomycin (100 pg/ml), and fungizone (2.5 pg/ml). Then they were adjusted to the final concentration of 12 X 106/ml in RPM1 1640 (GIBCO, Scotland) supplemented with penicillin, streptomycin, and fungizone at the same concentrations as in HBSS, and 1% glutamine (complete medium). 3. Mouse thyroid cell culture was performed as previously described (l-4). Briefly, thyroids were carefully dissected and minced with scissorsinto small pieces at 4°C in complete medium. The suspension was incubated with 1.5 mg/ml of collagenase (Boehringer-Mannheim, Mannheim, West Germany) in a shaking water bath at 37°C for 30 min. After washing (10 min, 250g) a second treatment with a new batch of collagenase was performed as described above. The cells were then washed and resuspended in complete medium supplemented with 5% fetal calf serum (FCS). About 2 X lo4 cells in 0.2 ml (mainly viable single thyroid cells as well as complete follicules) were incubated in a flat-bottomed Microtest II (Falcon 3042) tissue-culture dish. Incubation was performed at 37°C in a humidified atmosphere of 5% CO2 in air until used. They were used between Days 10 and 13 postculture because of both the optimal expression of H-2 K and D ends on TEC (9) and the smallest fibroblast or macrophage contamination (5% on Day 13) (1). 4. Primary syngeneic sensitization. Lymphoid cell suspensions, in complete medium without any sera becauseof their possible effect (2) were settled onto confluent monolayers of syngeneic TEC. Lymphoid cell suspension (0.2 ml) was cultured either on TEC or alone in order to determine the spontaneous thymidine incorporation of lymphocytes. After 2, 3,4, or 5 days of culture in a humidified incubator in 5% CO* in air, 1 &i of [‘Hlthymidine (sp act 5 &i/mmol) was added. Twelve hours later, the cells were harvested with a multiple automated sample harvester and the radioactivity incorporated by the cells was determined by liquid scintillation counting. In all cases,cultures were performed in quadruplicate and the mean cpm of thymidine incorporation in the quadruplicate calculated. 5. Expression ofthe results. The results were expressedas mean cpm of thymidine incorporation of sensitized lymphocytes minus mean cpm of thymidine incorporation of control lymphocytes alone (Acpm). As thymidine uptake varied quantitatively from experiment to experiment, lymphoproliferative responseswere compared to the lowest PSSgiven by C57BL/6 or BlO strains of mice, and expressedusing the index cpm of PSSor Expt ly - cpm of Expt ly alone cpm of PSSof B 10 or C57BL/6 - cpm of B 10 or C57BL/6 ly alone . In order to evaluate the levels of response, cpm of lymphocytes alone and PSS of BIO or C57BL/6 strains of mice are always cited. RESULTS Monolayers were prepared with thyroids from 2 1 inbred strains of mice. On Day 11 of culture, 0.25 X lo6 syngeneic lymphocytes were deposited onto the monolayers
389
SHORT COMMUNICATIONS
and the thymidine uptake was measured on Days 2, 3, 4, and 5 after this syngeneic stimulation. In the same experiment, at least seven different H-2 haplotypes were tested, always including as controls the good and poor responders to the EAT induced by Tg and adjuvant (CBA or Bl O.BR = H-2k and C57BL/6 or B 10 = H-2b). Additional control on other monolayers (i.e., syngeneic fibroblasts) was not performed because we previously demonstrated (2) that PSS on monolayer-s of TEC was strictly specific of thyroid antigens. As seenin Tables 1 and 2, a striking correlation between the syngeneic stimulation and the H-2 haplotype was found, with an optimal expression on Day 3 after stimulation. It must be noted that mice with different H-2 haplotypes were neither very sensitive nor completely resistant, but showed graded degreesof susceptibility. For instance, H-2k and H-2” (recombinant haplotype between k and d) lymphocytes were consistently excellent responders; H-2”, H-2’, and H-2” were good; while the other H-2 haplotypes responded poorly. PSS of H-2d haplotypes was difficult to classify since DBA/2 behaved like good responders while the congenic strain BlO.D2 responded poorly. In these experiments, H-2b strains were very poor responders and their PSS was used to determine background values in the calculation of stimulation indices. A very similar H-2 haplotype classification of good or poor responders to syngeneic sensitization on monolayers of TEC was obtained using congenic resistant strains of mice. However, among good responder H-2 haplotypes, decreased levels of stimuTABLE 1 Genetic Control of Primary Syngeneic Sensitization on Monolavers of TEC Days after stimulation Strains of TEC and lymphocytes AKR C3H CBA AC SJL DBA/Z BALB/c BALB/b C57BL/6 C57BL/6/PSS C57BL/6 spleen cells alone
H-2 haplotypes
2
H-2k H-2’ H-2’ H-2” H-2” H-2” H-2d H-2b H-2b
6.9” 16.6 25.5 11.4 8.6 6.7 1.1 1.7 1.0
18.9 k 0.5 21.1 f 2.7 26.5 + 4.6 13.7 + 2.6 9.4 ?I 0.9 8.7 +- 1.3 0.9 + 0.1 1.9 k 0.2 1.0 + 0.0
1253 f 104d 904 + 87
3
4
5
4.9 3.7 9.7 7.4 3.1 4.2 1.3 1.6 1.0
ND 0.9 0.3 1.5 1.7 1.2 ND 0.3 1.0
957 k 83
856 rf: 108
695 f 46
694 -e 64
488 + 52
453 f 34
4970b 5549 6969 3603 2472 2288 236 499 263
Note. Results are expressed as means f SEM of 2-4 determinations. ND, not determined. n Results are expressed as stimulation indices calculated as cpm of PSS of Expt ly - cpm of Expt ly alone cpm of PSS BlO or C57BL/6 - cpm of BlO or C57BL/6 ly alone . ’ Day +3 of PSS thymidine uptake is shown (Acpm). Ranges of [‘Hlthymidine for lymphocytes alone varies from 320 to 1350. ‘A is a recombinant haplotype between H-2k and H-2d. d Mean cpm of quadruplicates PSS of low-responder strains: C57BL/6 or BlO.
390
SHORT COMMUNICATIONS TABLE 2
Genetic Control of Primary Syngeneic Sensitization on Monolayers of TEC in Congenic Strains of Mice Days alter stimulation Strains of TEC and lymphocytes BIO.BR Bl0.A’ Bl0.S BlO.D2 Bl0.M BlO.WB Bl0.P Bl0.G Bl0.R III BlO.PL BlO.SM BlO CSlBL/lO/PSS C57BL/lO spleen cells alone
H-2 haplotype
2
H-2k H-2” H-2’ H-2* H-2’ H-2’ H-2P H-2q H-2’ H-2” H-2’ H-2b
15.5” 13.2 7.5 2.3 1.2 0.8 1.4 0.6 3.1 2.0 1.2 1.0
3 9.1 * 14.2 + 5.2 + 2.3 f 1.8 + 0.6 + 1.8 2 1.2 + 5.4 + 4.3 + 1.3 f 1.0 f
0.4 1.6 1.2 0.4 0.2 0.3 0.5 0.1 0.8 0.4 0.1 0.0
1998’ 2925 1071 414 371 124 371 247 1112 886 268 206
4
5
4.4 1.4 2.1 1.0 0.8 0.4 0.3 0.5 2.4 3.1 0.8 1.0
2.9 0.5 1.5 1.4 ND -
1601 f 136d
1130 + 121
829 f 125
935 f 140
1340 f 152
924 + 145
636 + 145
550 f
88
Note. Results are expressedas means k SEM of 2-4 determinations. See footnotes to Table 1.
lation were observed using congenic strains of mice rather than noncongenic ones (2 1.1 + 2.7 in CBA vs 15.5 + 0.4 in BlO.BR). This lower level of responseby congenic mouse strains has previously been reported by Rose et al. for EAT as being due to background ( 10). DISCUSSION The aim of this work was to determine if in vitro PSSon monolayers of TEC was under genetic control as previously described (5) for EAT in mice or spontaneous diseasesin rats (6), chickens (7) or humans (8). This question was raised because helper T cells were demonstrated in EAT in mice (10) and in this model of PSSon monolayers of TEC, T cells were shown to be the stimulated cells (2, 3). Regardless, these T cells could be prime candidates for fulfilling “in vitro” the “in vivo” action of helper T cells in EAT. Our study clearly demonstrates that this in vitro syngeneic sensitization on monolayers of TEC is under the genetic control of the MHC. However, a faint effect of the background on this stimulation was detected. More precisely, the quantitative difference was particularly strong in k-haplotype strains and especially in suboptimal conditions of culture. On Day +2, +3, or +4 of culture, PSS of CBA lymphocytes were respectively 4, 1.5, and 2 times higher than those of AKR mice, while PSS of C3H lymphocytes remains intermediate. In these three strains of k-haplotype mice since H-2 regions are identical, only the background could explain these differences. A similar result was found in EAT (10). It must be noted that in both PSS on TEC and EAT (where Tg is the stimulating antigen) the same H-2 haplotypes were found to be good or poor responders. The genetic control of PSSon TEC monolayers was detected as early as the second day of culture and persisted until Day 4. The optimal
SHORT COMMUNICATIONS
391
PSSwas obtained on the third day of culture. It was postulated that this rapid kinetic pattern of lymphocyte stimulation could be due to the fact that this immunological T-cell sensitization was due to a confluent monolayer of TBC used only when MHC antigens and thyroglobulin were optimally expressed (11) and not to a T-cell stimulation by non-T cells as described for autologous stimulation in mice (12) or humans (13). The good correlation found between the genetic control of EAT and PSSon monolayers of TEC strongly favors the hypothesis that the T cell implicated in PSSin vitro could be the helper T cell involved in EAT, as previously demonstrated by Beisel et al. ( 14) and Christadoss et al. ( 15). ACKNOWLEDGMENT We sincerely thank Ms. Janet Jacobson for reading the manuscript.
REFERENCES 1. Yeni, P., Michel-Bechet, M., Athouel-Haon, A. M., Fayet, G., and Charreire, J., In “Autoimmune Aspects of Endocrine Disorders” (A. Pinchera, D. Doniach, G. F. Fenzi, and L. Bachieri, Eds.), pp. 201-214. Academic Press,New York, 1980. 2. Yeni, P., and Charreire, J., Ceil. Immunol. 62, 3 13, 1981. 3. Charreire, J., Eur. J. Immunol. 5, 416, 1982. 4. Charreire, J., and Michel-Bechet, M., Eur. J. Immunol. 5, 421, 1982. 5. Vladutiu, A. O., and Rose, N. R., Science, 174, 1137, 1971. 6. Silverman, D. A., and Rose, N. R., J. Immunol. 114, 145, 1975. 7. Wick, G., Gundolf, R., and Hala, K., J. Immunogenet. 6, 177, 1979. 8. Irvine, J., In “Genetic Control of Autoimmune Disease? (N. R. Rose, P. Bigarzi, and N. Warner, Eds.), pp. 77-100, 1979. 9. Salamero, J., Michel-Bechet, M., and Charreire J., CR Acud. Sci. (Paris) 293, 745, 1981. 10. Rose, N. R., Kong, Y. M., Okayasu, I., Giraldo, A. A., Beisel, K., and Sundick, R. S., Transplant. Rev. 55, 299, 1981. 11. Salamero, J., Michel-B&het, M., and Charreire, J., Tissue Antigens, in press, 1983. 12. Glimcher, L. H., Steinberg, A. D., House, S. B., and Green, I., J. Immune/. 125, 1832, 1980. 13. Foumier, C., and Charreire, J., J. Immunol. 128, 2698, 1982. 14. Beisel, F. W., David, C. S., Giraldo, A. A., Kong, Y. M., and Rose N. R. Immunogenetics, 15, 427, 1982. 15. Christadoss, P., Kong, Y. M., Elrehewy, M., Rose, N. R., and David, C. S., In “Genetic Control of Autoimmune Disease” (N. R. Rose, P. E. Bigazzi, and N. L. Warner, Eds.), pp. 445-454. Elsevier/ North-Holland, Amsterdam/New York, 1978.
IMMUNOLOGY
78, 387-39 I (1983)
Syngeneic Sensitization of Mouse Lymphocytes Monolayers of Thyroid Epithelial Cells IV. Correlation
on
with H-2 Haplotypes
JEANSALAMEROANDJEANNINECHARREIRE Inserm U 25, Hspital Necker, 161 rue de S&es,
75730 Paris C&dex 15, France
Received December 21, 1982; accepted March 9, 1983 In vitro primary syngeneicsensitization on monolayer of thyroid epithelial cells was performed with 21 inbred strains of mice representing 11 original H-2 haplotypes. Significant differences in the proliferative responses,assessedby thymidine uptake, were found to be related to the major h&compatibility complex haplotype. This result was further confirmed using congenic resistant strains of mice. In comparison with the experimental autoimmune thyroiditis induced by syngeneic thyroglobulin and adjuvant, primary syngeneic sensitization on monolayers of thyroid epithelial cells appeared to be under the same genetic control (H-2k strains being good responders, while H-2b mice are poor responders).
INTRODUCTION During “in vitro” primary syngeneic sensitization (PSS)’ on monolayers of thyroid epithelial cells (1, 2), we demonstrated that T lymphocytes were stimulated (3) and able to induce thyroiditis when injected into normal syngeneic recipients (4). The evidence for immunological sensitization is provided by both the specificity of the stimulation directed only against syngeneic thyroid epithelial cells (TEC) and the production of memory cells detected by a characteristic secondary stimulation on syngeneic monolayers of TEC (2). Using this simple and objective model, we investigated if this stimulation was under genetic control as described for murine experimental autoimmune thyroiditis (5) in which the animals were immunized with thyroglobulin (Tg) and complete Freund’s adjuvant or spontaneous diseasesin rats (6) chickens (7), and humans (8). MATERIALS
AND METHODS
I. Animals. The mice strains AKR (H-2k), C3H (H-2’), CBA (H-2k), A (H-29, SJL (H-2’) DBA/2 (H-2d), BALB/c(H-2d), BALB/b (H-2b), and C57BL/6 (H-2b) were purchased from the Centre de Selection et d’Elevage des Animaux de Laboratoire, CNRS, Orleans la Source, France. The mice strains of the BlO background, BlO.BR ’ Abbreviations used: EAT, experimental autoimmune thyroiditis; FCS, fetal calf serum; HBSS, Hanks’ balanced salt solution; MHC, major histocompatibility complex; NMS, normal mouse serum; ESS,primary syngeneic sensitization; TEC, thyroid epithelial cells; Tg, thyroglobulin. 387 0008-8749/83 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.
388
SHORT
COMMUNICATIONS
(I-I-2k), BIO.A (H-2”), BIOS (H-2S), BlO.D2 (H-2d), BIO.M (H-21, BlO.WB (H-2’), Bl0.P (H-2P), BIO.G (H-2’), BIO.R III (H-23, BlO.PL (H-2U), BlOSM (H-2’), and B 10 (H-2b), were donated by ProfessorColombani, Hopital Saint Louis, Paris, France. All these animals were used between 8 and 16 weeks of age. 2. Lymphoid cell suspensions. Spleen cells were aseptically removed and homogenized in a glasstube with a loose-fitting Teflon pestle. Cell suspensionswere washed twice in Hanks’ balanced salt solution (HBSS) supplemented with penicillin (100 U/ ml), streptomycin (100 pg/ml), and fungizone (2.5 pg/ml). Then they were adjusted to the final concentration of 12 X 106/ml in RPM1 1640 (GIBCO, Scotland) supplemented with penicillin, streptomycin, and fungizone at the same concentrations as in HBSS, and 1% glutamine (complete medium). 3. Mouse thyroid cell culture was performed as previously described (l-4). Briefly, thyroids were carefully dissected and minced with scissorsinto small pieces at 4°C in complete medium. The suspension was incubated with 1.5 mg/ml of collagenase (Boehringer-Mannheim, Mannheim, West Germany) in a shaking water bath at 37°C for 30 min. After washing (10 min, 250g) a second treatment with a new batch of collagenase was performed as described above. The cells were then washed and resuspended in complete medium supplemented with 5% fetal calf serum (FCS). About 2 X lo4 cells in 0.2 ml (mainly viable single thyroid cells as well as complete follicules) were incubated in a flat-bottomed Microtest II (Falcon 3042) tissue-culture dish. Incubation was performed at 37°C in a humidified atmosphere of 5% CO2 in air until used. They were used between Days 10 and 13 postculture because of both the optimal expression of H-2 K and D ends on TEC (9) and the smallest fibroblast or macrophage contamination (5% on Day 13) (1). 4. Primary syngeneic sensitization. Lymphoid cell suspensions, in complete medium without any sera becauseof their possible effect (2) were settled onto confluent monolayers of syngeneic TEC. Lymphoid cell suspension (0.2 ml) was cultured either on TEC or alone in order to determine the spontaneous thymidine incorporation of lymphocytes. After 2, 3,4, or 5 days of culture in a humidified incubator in 5% CO* in air, 1 &i of [‘Hlthymidine (sp act 5 &i/mmol) was added. Twelve hours later, the cells were harvested with a multiple automated sample harvester and the radioactivity incorporated by the cells was determined by liquid scintillation counting. In all cases,cultures were performed in quadruplicate and the mean cpm of thymidine incorporation in the quadruplicate calculated. 5. Expression ofthe results. The results were expressedas mean cpm of thymidine incorporation of sensitized lymphocytes minus mean cpm of thymidine incorporation of control lymphocytes alone (Acpm). As thymidine uptake varied quantitatively from experiment to experiment, lymphoproliferative responseswere compared to the lowest PSSgiven by C57BL/6 or BlO strains of mice, and expressedusing the index cpm of PSSor Expt ly - cpm of Expt ly alone cpm of PSSof B 10 or C57BL/6 - cpm of B 10 or C57BL/6 ly alone . In order to evaluate the levels of response, cpm of lymphocytes alone and PSS of BIO or C57BL/6 strains of mice are always cited. RESULTS Monolayers were prepared with thyroids from 2 1 inbred strains of mice. On Day 11 of culture, 0.25 X lo6 syngeneic lymphocytes were deposited onto the monolayers
389
SHORT COMMUNICATIONS
and the thymidine uptake was measured on Days 2, 3, 4, and 5 after this syngeneic stimulation. In the same experiment, at least seven different H-2 haplotypes were tested, always including as controls the good and poor responders to the EAT induced by Tg and adjuvant (CBA or Bl O.BR = H-2k and C57BL/6 or B 10 = H-2b). Additional control on other monolayers (i.e., syngeneic fibroblasts) was not performed because we previously demonstrated (2) that PSS on monolayer-s of TEC was strictly specific of thyroid antigens. As seenin Tables 1 and 2, a striking correlation between the syngeneic stimulation and the H-2 haplotype was found, with an optimal expression on Day 3 after stimulation. It must be noted that mice with different H-2 haplotypes were neither very sensitive nor completely resistant, but showed graded degreesof susceptibility. For instance, H-2k and H-2” (recombinant haplotype between k and d) lymphocytes were consistently excellent responders; H-2”, H-2’, and H-2” were good; while the other H-2 haplotypes responded poorly. PSS of H-2d haplotypes was difficult to classify since DBA/2 behaved like good responders while the congenic strain BlO.D2 responded poorly. In these experiments, H-2b strains were very poor responders and their PSS was used to determine background values in the calculation of stimulation indices. A very similar H-2 haplotype classification of good or poor responders to syngeneic sensitization on monolayers of TEC was obtained using congenic resistant strains of mice. However, among good responder H-2 haplotypes, decreased levels of stimuTABLE 1 Genetic Control of Primary Syngeneic Sensitization on Monolavers of TEC Days after stimulation Strains of TEC and lymphocytes AKR C3H CBA AC SJL DBA/Z BALB/c BALB/b C57BL/6 C57BL/6/PSS C57BL/6 spleen cells alone
H-2 haplotypes
2
H-2k H-2’ H-2’ H-2” H-2” H-2” H-2d H-2b H-2b
6.9” 16.6 25.5 11.4 8.6 6.7 1.1 1.7 1.0
18.9 k 0.5 21.1 f 2.7 26.5 + 4.6 13.7 + 2.6 9.4 ?I 0.9 8.7 +- 1.3 0.9 + 0.1 1.9 k 0.2 1.0 + 0.0
1253 f 104d 904 + 87
3
4
5
4.9 3.7 9.7 7.4 3.1 4.2 1.3 1.6 1.0
ND 0.9 0.3 1.5 1.7 1.2 ND 0.3 1.0
957 k 83
856 rf: 108
695 f 46
694 -e 64
488 + 52
453 f 34
4970b 5549 6969 3603 2472 2288 236 499 263
Note. Results are expressed as means f SEM of 2-4 determinations. ND, not determined. n Results are expressed as stimulation indices calculated as cpm of PSS of Expt ly - cpm of Expt ly alone cpm of PSS BlO or C57BL/6 - cpm of BlO or C57BL/6 ly alone . ’ Day +3 of PSS thymidine uptake is shown (Acpm). Ranges of [‘Hlthymidine for lymphocytes alone varies from 320 to 1350. ‘A is a recombinant haplotype between H-2k and H-2d. d Mean cpm of quadruplicates PSS of low-responder strains: C57BL/6 or BlO.
390
SHORT COMMUNICATIONS TABLE 2
Genetic Control of Primary Syngeneic Sensitization on Monolayers of TEC in Congenic Strains of Mice Days alter stimulation Strains of TEC and lymphocytes BIO.BR Bl0.A’ Bl0.S BlO.D2 Bl0.M BlO.WB Bl0.P Bl0.G Bl0.R III BlO.PL BlO.SM BlO CSlBL/lO/PSS C57BL/lO spleen cells alone
H-2 haplotype
2
H-2k H-2” H-2’ H-2* H-2’ H-2’ H-2P H-2q H-2’ H-2” H-2’ H-2b
15.5” 13.2 7.5 2.3 1.2 0.8 1.4 0.6 3.1 2.0 1.2 1.0
3 9.1 * 14.2 + 5.2 + 2.3 f 1.8 + 0.6 + 1.8 2 1.2 + 5.4 + 4.3 + 1.3 f 1.0 f
0.4 1.6 1.2 0.4 0.2 0.3 0.5 0.1 0.8 0.4 0.1 0.0
1998’ 2925 1071 414 371 124 371 247 1112 886 268 206
4
5
4.4 1.4 2.1 1.0 0.8 0.4 0.3 0.5 2.4 3.1 0.8 1.0
2.9 0.5 1.5 1.4 ND -
1601 f 136d
1130 + 121
829 f 125
935 f 140
1340 f 152
924 + 145
636 + 145
550 f
88
Note. Results are expressedas means k SEM of 2-4 determinations. See footnotes to Table 1.
lation were observed using congenic strains of mice rather than noncongenic ones (2 1.1 + 2.7 in CBA vs 15.5 + 0.4 in BlO.BR). This lower level of responseby congenic mouse strains has previously been reported by Rose et al. for EAT as being due to background ( 10). DISCUSSION The aim of this work was to determine if in vitro PSSon monolayers of TEC was under genetic control as previously described (5) for EAT in mice or spontaneous diseasesin rats (6), chickens (7) or humans (8). This question was raised because helper T cells were demonstrated in EAT in mice (10) and in this model of PSSon monolayers of TEC, T cells were shown to be the stimulated cells (2, 3). Regardless, these T cells could be prime candidates for fulfilling “in vitro” the “in vivo” action of helper T cells in EAT. Our study clearly demonstrates that this in vitro syngeneic sensitization on monolayers of TEC is under the genetic control of the MHC. However, a faint effect of the background on this stimulation was detected. More precisely, the quantitative difference was particularly strong in k-haplotype strains and especially in suboptimal conditions of culture. On Day +2, +3, or +4 of culture, PSS of CBA lymphocytes were respectively 4, 1.5, and 2 times higher than those of AKR mice, while PSS of C3H lymphocytes remains intermediate. In these three strains of k-haplotype mice since H-2 regions are identical, only the background could explain these differences. A similar result was found in EAT (10). It must be noted that in both PSS on TEC and EAT (where Tg is the stimulating antigen) the same H-2 haplotypes were found to be good or poor responders. The genetic control of PSSon TEC monolayers was detected as early as the second day of culture and persisted until Day 4. The optimal
SHORT COMMUNICATIONS
391
PSSwas obtained on the third day of culture. It was postulated that this rapid kinetic pattern of lymphocyte stimulation could be due to the fact that this immunological T-cell sensitization was due to a confluent monolayer of TBC used only when MHC antigens and thyroglobulin were optimally expressed (11) and not to a T-cell stimulation by non-T cells as described for autologous stimulation in mice (12) or humans (13). The good correlation found between the genetic control of EAT and PSSon monolayers of TEC strongly favors the hypothesis that the T cell implicated in PSSin vitro could be the helper T cell involved in EAT, as previously demonstrated by Beisel et al. ( 14) and Christadoss et al. ( 15). ACKNOWLEDGMENT We sincerely thank Ms. Janet Jacobson for reading the manuscript.
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