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Neonatal tolerance induction in the thymus to MHC-class II-associated antigens
CELLULAR
IMMUNOLOGY
103,l l- 18 ( 1986)
Neonatal Tolerance Induction in the Thymus to MHC-Class II-Associated Antigens II. Significance
of MHC Antigens in Anti-MIS Tolerance
MASAMICHI HOSONO AND YOSHIMOTO KATSURA Department oJBacteriology and Serology, Chest Disease Research Institute, Kyoto University, Kyoto 606, Japan Received January 17,1986; acceptedJune 5,1986 Specificity of anti-Mlsa tolerance induced in BALB/c (H-2d, Mlsb) neonates was investigated by a pop&al lymph node (PLN)-swelling assay for the local graft-versus-host (GVH) reaction by injecting tolerant thymus cells into the footpads of several types of Fl hybrid mice. When thymus cells were obtained from l-week-old normal BALB/c, they evoked enlargement of PLNs of (BALB/c X DBA/Z)F 1(H-2d, Mlsb”) [CDF 1] recipients and of other hybrid recipients, heterozygous in Ml.+csdalleles, irrespective of the major histocompatibility complex (MHC) haplotypes. The same thymus cells did not causethe responsein MHC-heterozygous Fl hybrids when the hybrids were homozygous in Mlsb, identical with BALB/c mice. Therefore, the PLN response to Mls antigens, known to be closely associated with MHC-class II antigens, was not directed to the class II antigens themselves. This enabled us to examine the effects of MHC on tolerance induction to the Mls antigens. When BALB/c neonates were injected with CDFl bone marrow cells, complete tolerance to M~s’-H-~~antigens of CDFl cells was induced in the thymus, while responsivenessto Mls” antigens in the context of H-2’ and H-2b antigens, was not alEcted. This indicates MHC-restriction of neonatal tolerance to Mls antigens. Furthermore, when Mls and H-2-heterozygous (BALB/c X AKR)Fl (H-2W, Mlsb”) bone marrow cells served as the tolerogen, thymus cells of BALB/c neonates were also tolerized to Mls’-H-2’ antigens as well asto M~s*-H-~~antigens, which suggeststhe involvement of MHC, probably classII antigens of tolerance-inducing cells. 0 1986 Academic PITS, Inc.
INTRODUCTION Neonatal injection of major histocompatibility complex (MHC)-allogeneic and semiallogeneic hematopoietic cells induced unresponsiveness to the corresponding antigens asjudged at adulthood in in vivo (1,2) and in vitro (3-5) assaysystems. The results obtained by the tolerance induction method developed originally as a model of self tolerance, may not be extended to whole sets of self antigens, since recent reports suggest that the responses to allo-MHC antigens differ from those to nonMHC, minor antigens in terms of T cell subpopulations (6,7), physiology of antigenpresenting cells (8), and tolerance inducibility (9-l 1). Previously (12), we suggestedthat different mechanisms are involved in neonatal tolerance induction to allo-MHC antigens and non-MHC antigens and that immunological tolerance to the latter antigens to which the T-cell response is restricted to selfMHC antigens may provide a good model for self tolerance to non-MHC, conven11 0008-8749186$3.00 Copyrisht8 1986 by Academic
F’ms, Inc. AII rigbis of rcpraduction in any form resmed.
12
HOSONO AND KATSURA
tional antigens, as reported by others using different experimental systems ( 13-l 8). When we consider ontogenetic development of self tolerance, MHC-class II restriction of tolerance induction is of interest as the mechanism for negative selection of T cells committed to self antigens, since expression of Ia antigens is primarily localized in the thymus (19-24). Intravenous injection of Mls-semiallogeneic bone marrow cells readily induced neonatal tolerance in thymic T cells directing to the Mls antigens (12), to which Tcell activation responsesare closely associatedwith Ia antigens on the stimulator cells (25-27). In this respect, we examined whether the anti-Mullstolerance induction in newborn thymus is affected by the MHC haplotype of tolerance-inducing bone marrow cells. MATERIALS AND METHODS Most materials, methods, and experimental schedules employed in this study were as described in detail previously ( 12). Mice used here were BALB/c (H-2d, Mlsb), DBA/2 (H-2d, MIS’), BlO.BR (H-2k, Mlsb), C3H/He (H-2k, Mls”), and C57BL/6 (H-2b, Mlsb) purchased from the Shizuoka Agricultural Cooperative Association for Laboratory Animals (Shizuoka). AKR/J (H-2’, Ml?) and CBA/J (H-2k, Mlsd) mice were donated by Dr. S. Muramatsu, Faculty of Science, Kyoto University. Fl hybrids between these strains of mice were produced in our animal facility and used at 3 to 5 months of age. Tolerance was induced in BALB/c neonates (24 hr after birth) by intravenous injection of semiallogeneic bone marrow cells (2 X 10’ in 50 ~1). One week later, the mice were killed and their thymus cells were tested in vivo for their activity to cause the local graft-versus-host reaction (GVHR) by injecting thymus cells (2 X 10’ in 25 ~1)into the footpads of the Fl hybrids. Further 1 week later, popliteal lymph nodes (PLN) of each Fl hybrid recipient were removed and weighed. As negative controls, the medium was injected. The degree of PLN enlargement was expressed as lymph node (LN)-swelling index (LN index = PLN weight of cell-injected group/PLN weight of negative control group). Percentage of tolerance was calculated as follows: percentage tolerance = [ 1 - (mean PLN weight of tolerant group - mean PLN weight of negative control)/(mean PLN weight of non-tolerized group - mean PLN weight of negative control)] X 100, as previously described ( 12). RESULTS
Ontogenyof Thymus Cells Reactiveto Allo-MS and MHC-Class II Antigens Figure 1 shows the developmental profiles in ontogeny of anti-Mls and H-2 reactivities of thymus cells. Injection of thymus cells of BALB/c (H-2d, Mlsb) mice of various agesinto the footpads of (BALB/c X DBA/2)Fl (H-2d, Mlsb@) mice and (BALB/c X BlO.BR)Fl (H-2d/@,Mlsb) mice caused popliteal lymph node (PLN) enlargement as a result of a graft-versus-host reaction (GVHR) ( 12): the disparity in Mls and H-2 haplotypes in comparison with BALB/c mice is indicated in a circle. As shown in this figure, the activity to cause the anti-Ml!? GVHR was already appreciable at 24 hr of age, and reached plateau values by 1 week of age. In contrast, anti-H-2k activity was not detectable during the first 2 weeks of age. The delay in reaching the plateau value and low level of anti-allo-class II response, as compared with anti-Mls responsiveness,
MHC-ASSOCIATION
OF ANTI-Mls TOLERANCE
2
1
0
3
Weeks after
13
4
birth
FIG. I. Ontogeny of anti-Mls and Ia activity of BALB/c thymus cells. Thymus cells prepared from individual BALB/c (H-2d, Mlsb) mice of various agesas indicated on abscissa,were injected into the footpads of (BALB/c X DBA/2)Fl (H-2d, Mlsbl@)(0) and of (BALB/c X BlO.BR)Fl (H-2‘@, Mlsb) (0) mice for the local GVHR. Each symbol indicates the activity of each thymus. Horizontal striped bar represents + 1 SE of the mean of control groups ( 1.00 + 0.05 and 1.OOf 0.09 for anti-Mls’ and anti-I$ activity, respectively).
may reflect not only the different size of responding cell population but also the delayed maturation of anti-allo-class II activity. Unresponsiveness to allo-H-2 antigens of l-week-old BALB/c thymus cells was also observed for H-2b haplotypes of antigens as tested in (BALB/c X C5’7BL/6)Fl (H-2 dti Mlsb) hosts (Fig. 2). The lack of activity in l-week-old thymus cells was not attributable to the number of inoculated cells since fewer thymus cells showed no activity.
Demonstration of Anti-Mls Activity but not of Anti-allo-Class II Activity in I-WeekOld Thymus Anti-Mlsqd activity in 1-week-old BALB/c thymus cells were investigated by means of the local GVHR in various types of Fl hybrid recipients. These recipients were either homozygous or heterozygous for H-2d and/or Mlsb haplotypes, so that the involvement of host-versus-graft reaction could be excluded in the PLN-swelling assay.
4r
_I 0625
025
05
1.0
20
Na of thymus cells (x10’) FIG. 2. Cell dose effect on anti-Mls and H-2 GVHR. Various numbers of thymus cells of l-week-old BALB/c mice, as indicated on abscissa,were injected into (BALB/c X DBA/Z)Fl (H-2d, Ml@‘@)mice (0) and (BALB/c X C57BL/6)Fl (H-2@, Mlsb) mice (0) for assessinganti-Mls” and Iab activity, reqectively. Standard errors of the mean are represented by vertical bars, or omitted when they were smaller than the symbol marks. The horizontal striped bars represent f 1 SE of the mean of the control groups which received injections of medium (1.OO+ 0.12 and 1.oO+ 0.04 for anti-Mb’ and anti-Iab activity, respectively).
14
HOSONO AND RATSURA TABLE I Anti-Mls GVH Activity of l-Week-Old BALB/c Thymus Cells” Disparity in Group
Fl hybrid recipients (BALB/c X DBA/Z)F 1 (BALB/c X C57BL/6)Fl (DBA/Z X C57BL/6)Fl (BALB/c X BlO.BR)Fl (DBA/Z X BlO.BR)Fl (BALB/c X ARR)Fl (BALB/c X CBA/J)FI (BALB/c X C3H/He)Fl
H-2 -
b b k k k k k
Mls
LN index & SE (n)
a a a
3.23 +-0.19 (23) 1.15~0.05(11) 3.29 f 0.29 (7) 1.53+0.09(17) 3.22 + 0.37 (8) 2.78 f 0.11 (7) 2.56 + 0.24 (23) 2.04 + 0.16 (5)
Ii C
’ GVHR-inducing activity of 1-week-old, normal BALB/c thymus cells is expressed as LN index + SE in the PLN-swelling assayby injecting thymus cells into the footpads of Fl hybrid mice. Figures in parenthesesindicate the number of PLNs tested.
As summarized in Table 1, the newborn thymus cells caused PLN enlargement when recipients were heterozygous in Mls”, Mls”, and Mlsd, irrespective of MHC haplotypes (Groups 1, 3, and 5-8): stronger responses to Mlsasdantigens than to Mls” antigens was in agreement with the in vitro results in the mixed lymphocytes reaction (28, 29). On the other hand, they had no effect on the Mls-identical, MHC-heterozygous recipients (Groups 2 and 4). Thus, l-week-old thymus cells appear to be responsive to allo-Mls antigens but not to allo-class II antigens.
MHC Restriction ofAnti-Mls ToleranceInduction As shown in the top groups of experiments A and B of Fig. 3, the thymus cells became tolerant (80-90s) to M~s*-H-~~ antigens and showed only the background level response of medium controls. The same samples of thymus cells, on the other hand, evoked stron GVHR in (BALB/c X AKR)Fl (H-2d/@,Mlsb@) and (C57BL/6 X DBA/2)F 1 (H-2 8b’d Mlsb@) hosts, both of which express Mls” antigens and allogeneic H-2 antigens, and in (BALB/c X CBA/J)Fl (H-2d’@, Mlsb@) hosts, expressing M~s~-H-~~““antigens. These results were not attributed to reactivity of the thymus cells to allogeneic H-2 antigens themselves, since they had no effect on (BALB/c X BlO.BR)Fl (H-2d@, Mlsb) or (BALB/c X C57BL/6)Fl (H-2d’@,Mlsb) hosts.
MHC Haplotypes of Tolerance-InducingCells in Anti-k% Tolerance We examined whether the MHC haplotype of tolerance-inducing cell donors is responsible for the MHC restriction in anti-Mls neonatal tolerance, using MHC-heterozygous (BALB/c X AKR)F 1 (H-2d’@,Mlsb@) bone marrow cells as tolerogens. As shown in Table 2, the percentage of tolerance for the corresponding M~s~-H-~~~test antigens was higher than that for M~s”-H-~~antigens of (BALB/c X DBA/2)Fl mice (Groups 1 and 2), suggesting that potential responsiveness to M~s”-H-~~ antigens, in addition to M~s~-H-~~antigens, is also suppressed.Such hyporesponsiveness is again specific to the Mls antigens in association with MHC haplotypes of the tolerance-
MHC-ASSOCIATION Hybrid recipients (H-2 1 MIS) W A (BALBk xDbv2P
OF ANTI-Mb
15
LN index N
.b@)
T
(BA!eB/c x AKR)R I dC3.W)
7
M&cx~BA)FI
:
‘BAI.~
b” FlO BR)Fl
;
‘“;“;,B$
; ~57WFt
;
(d
TOLERANCE
FIG. 3. Specificity of neonatally induced tolerance to the Mls antigen. BALB/c (H-2d, Mlsb) neonates were injected with bone marrow cells of (BALB/c X DBA/2)Fl (H-2d, Mlsb’@)mice. Thymus cells of the tolerized BALB/c mice were injected into the footpads of Fl hybrids homozygous or heterozygous in H-2d and Mlsb as indicated (T, w). The same numbers of thymus cells of age-matched, normal mice were used as positive controls (N, 0). One week after the footpad injection, the Fl mice were killed and their popliteal lymph nodes were weighed for the GVHR. The horizontal column with bar represents the mean LN index with + 1 SE and the vertical striped bar represents + 1 SE of the mean of negative control group receiving injections of medium. In experiment B, the bone marrow cells used as tolerogens, were pretreated with anti-Thy. 1 monoclonal antibody plus complement to remove T cells, if any, before injection.
inducing cells, the GVHR-inducibility (Group 3).
to M~s”-H-~~ antigens being not affected
Antigen Specificity ofAnti-Mls” Tolerancein the Thymus BALB/c neonates again were tolerized with (BALB/c X AKR)Fl bone marrow cells, and the antigen specificity of anti-Mls” tolerance in the thymus was tested. Table 3 shows the results. The degree of tolerance to M~s~-H-~~* antigens and M~s”-H-~~ antigens were 75 and 5 l%, respectively (Groups 1 and 2), confirming the previous experimental results. The same sample of thymus cells showed 49% cross tolerance to M~s~-H-~~~antigens of (BALB/c X CBA/J)Fl mice (Group 3), and normal activity to M~s”-H-~~~ antigens of (BALB/c X C3H/He)Fl mice (Group 4). DISCUSSION To find a clue for understanding the mechanisms of self tolerance in normally developing thymus cells contributing in response to MHC-class II-associated antigens, the Mls antigens seemed to be appropriate antigens, because of their strong immunogenicity in primary response in a fashion of self-class II restriction (25-27) not only for peripheral mature T cells but also for thymus cells ( 11, 12). Furthermore, neonatal tolerance to this antigen is relatively easily induced in thymus and spleen T cells within 1 week and continues for at least 4 months after birth. As we showed, the induction of neonatal tolerance to Mls antigens seemsto be associated with MHC-
16
HOSONO AND KATSURA TABLE
2
Restriction of Anti-Ml9 Tolerance Induction to the MHC Haplotype of Tolerance-Inducing Bone Marrow Cells” stateof Fl
hybrid recipients[H-2,
1 (BALB/c
X AKR)FI
[d/B,
Mls] b/@]
2 (BALB/c
X DBA/2)Fl
[d, b/@]
3 (DBA/Z
X C57BL/6)Fl
[d/B,
4 (BALB/c
X C57BL/6)Fl
[d/Q,
a/b] b]
BALB/c mice
LN index + SE(n)
Nor. Tol. Nor. Tol. Nor. Tol. Nor. Tol.
2.75 + 0.15 (4) 1.42+0.18(4) 3.56 + 0.20 (5) 2.28 + 0.16 (5) 3.77 f 0.56 (3) 3.15*0.16(3) 1.15 kO.lO(4) 1.53 f 0.04 (3)
P -
96 To1e.ranc-e -
0.02 -
77 -
0.01 -
50 -
0.70 -
Nontol.’ -
0.06
Nontol.
a BALB/c [H-2d, Mlsb] neonates received bone marrow cells of (BALB/c X AKR)Fl [H-2@, MlsWO] mice. One week later, thymus cells of the cell-injected BALB/c mice (Tol.) were routinely tested for GVHR inducibility in various types of Fl hybrid mice.Thymescellsof uninjectedlittermatesor age-matchedmicewereusedaspositivecontrols(Nor.). b Nontol. = nontolerant (P > 0.05).
haplotypes of the tolerance-inducing bone marrow cells, confirming the findings that tolerance to non-MHC antigens is restricted to self-MHC antigens (13-l 8). Together with the fact that Ia antigens appear first in the thymus (19-24), MHC restriction of class II-reactive T-cell tolerance predicts that the thymus is a site of clonal elimination of the classII-reactive T cells committed to self antigens, especially at an early stage of development when there are few Ia+ cells in periphery, and a site of Ia-expression may be closely related to the site where tolerance induction of the class II-reactive T cells is initiated. In this sense,tolerance to peripheral, non-MHC antigens would occur after their entrance into the thymus and in association with Ia antigens of thymic non-lymphoid cells (30). On the other hand, it is reasonable to assume that the extrathymic presence of Ia antigen-bearing cells in the periphery, in unphysiological or experimental conditions, aEectsT-cell repertoire generation, if Tcell receptors are prethymically expressed,as suggestedelsewhere ( 11,3 l-33). TABLE 3 Antigen Specificity of Anti-Ml9 Tolerance in the Thymus” stateof BALBjc Fl
hybrid recipients[H-2, Mls]
1 (BALB/cXAKR)Fl 2 (BALB/c 3
X DBA/Z)Fl
[d/@,b/@] [d, b/ @]
(BAWc X CWJW Id/ @ , b/ @ 1
4 (BALB/c
X C3H/He)Fl
[d/ @ , b/Q]
mice
LN index k
SE (n)
P
w Tolerance -
Nor.
4.92 f 0.82 (5)
-
Tol. Nor.
2.00 f 0.36 (4) 3.83 f 0.21 (5)
0.01 -
75 -
Nor.
Tol.
2.36 f O.ll(6) 2.77 + 0.1 I (5)
0.01 -
51 -
Tol. Nor. Tol.
1.91 f 0.07 (4) 2.04 f 0.16 (5) 1.9OiO.11 (6)
0.01 0.66
49 Nontol.b
’ Experimental system and schedule were the same as those employed in Table 2, except for types ofF1 hybrid recipients used for GVHR. b Nontol. = nontolerant (P > 0.05).
MHC-ASSOCIATION
OF ANTI-Mls TOLERANCE
17
However, this may not be the casefor the Mls antigens, since Morrissey et al. (11) showed that, in radiation chimera experiments, tolerance to the antigens was only induced intrathymically. Therefore, in our model of neonatal tolerance, entrance into the thymus of donor stem cell-originated non-lymphocytes, Ia positive, must be a requisite for the negative selection process. Such dependence of anti-Mls-tolerance induction on Ia-bearing non-lymphocytes in developing thymus may provide a useful model for the analysis of biological significance of intrathymic expression of Ia antigens in early stageof ontogeny, since the degree of Mls-specific tolerance depends on the number of tolerance-inducing cells injected and on MHC (Mls)-gene dosage, MHC (Mls)-homozygous tolerogen being twice as tolerogenic as MHC (Mls)-heterozygous tolerogen ( 12). Although we have tentatively referred to the Mls antigen as a non-MHC conventional antigen, as has been indicated by Berumen et al. (34,35) and Click et al. (36), the identity and structure of the Mls antigen is controversial, since the molecular nature of the antigen has not yet been defined. Janeway et al. (38) recently proposed that the Mls-gene functions in stabilizing T-cell-Ia antigen interaction rather than in producing a particular immunogenic component. According to Janeway’s model, Mls-gene product(s) on Ia-bearing, antigen-presenting cells (APC) stabilize the T-cell-APC interaction, so that preferential stimulation of low affinity self-class II-reactive T cells would occur, as in autologous mixed lymphocyte reaction. This would explain the high frequency of Mls-reactive T cells (39, 40) and probably as a result, the early appearance of anti-Mls reactivity in the thymus. As mentioned above, neonatal tolerance to “Mls antigens” is closely associated with MHC-haplotypes of tolerogen and tolerance to M~s”-H-~~‘~ antigens showed also reduced responsiveness to M~s~-H-~~~antigens. Thus, the model could explain most of our experimental observations. However, it should be noted that in preliminary experiments with the local HVG assaysystem (BALB/c X CBA/J)Fl (H2d/k, Mlsbld) mice responded to M~s”-H-~~‘~ antigens of (DBA/2 X BlO.BR)Fl (H-2dF, Ml@‘) mice, while no response was observed in the reciprocal donor-recipient combinations, suggestingthat T-cell population directing to the Mlsd antigens are not identical to those to the Mls” antigens, and that tolerance to M~s”-H-~~~ antigens did not affect the responsivenessto M~s”-H-~~~antigens, as reported here, suggesting that the Mls”-gene function differs from that of Mls”-gene. These data are in agreement with the concept of polymorphism in the Mls-gene function. Studies on the specificity of tolerance to MlsdYcantigens, in addition to Mls” antigens, are in progress. ACKNOWLEDGMENT The work was supported by a grant for the Shimizu Foundation Research.
REFERENCES 1. Billingham, R. E., Brent, L., and Medawar, P. B., Phil. Trans. R. Sot. London B 239,357,1956. 2. Stiobrocic, V., Eur. J. Immunol. I,3 13, 1971. 3. Gorczynski, R. M., and MacRae, S., J. Immunol. 122,737,1979. 4. Nossal, G. J. V., and Pike, B. L., Proc. Natl. Acad. Sci. USA 78,3844,198 1. 5. Feng, H. M., Glassbrook, A. L., Engers, H. D., and Louis, J. A., J. Zmmunol. 131,2165, 1983. 6. Hurme, M., J. Exp. Med. 149,290, 1979. 7. Sihvola, M., and Hurme, M., J. Immunol. 130, 1077, 1983. 8. Finnegan, A., Needleman, B. A., and Hades, R. J., J. Zmmunol. 134,2960, 1985.
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HOSONO AND KATSURA
9. Streilein, J. W., and Klein, J., Transpl. Proc. 11,732, 1979. 10. McCarthy, S. A., and Bach, F. H., J. Zmmunol. 131, 1670, 1983. 11. Morrissey, P. J., Bradley, D., Sharrow, S. O., and Singer, A., J. Exp. Med. 158,365, 1983. 12. Hosono, M., Kina, T., Hosokawa, T., and Katsura, Y., Cell. Zmmunol. 103,1,1986. 13. DosReis, G. A., and Shevach, E. M.,J. Exp. Med. 157, 1287, 1983. 14. Groves, E. S., and Singer, A., .I. Exp. Med. 158, 1483, 1983. 15. Lamb, J. R., and Feldmann, M., Nature (London) 308,72, 1984. 16. Lowy, A., Drebin, J. A., Monroe, J. G., Granstein, R. D., and Greene, M. I., Nature308,373, 1984. 17. Matzinger, P., Zamoyska, R., and Waldmann, H., Nature (London) 308,738, 1984. 18. Rammensee, H. G., and Bevan, M. J., Nature (London) 308,741,1984. 19. Jenkinson, E. J., Owen, J. J. T., and Aspinall, R., Nature(London) 284, 177, 1980. 20. Lu, C. Y., Beller, D. I., and Unanue, E. R., Proc. Natl. Acad. Sci. USA 77, 1597, 1980. 21. Natali, P. G., Nicotra, M. R., Giacomini, P., Pellegrino, M. A., and Freone, S., Immunogenetics 14, 359,198l. 22. Kyewski, B. A., Rouse, R. V., and Kaplan, H. S., Proc. Natl. Acad. Sci. USA 79,5646,1982. 23. Robinson, J. H., J. Zmmunol. 130,1592, 1983. 24. Hosono, M., Katsura, Y., and Muramatsu, S., ZmmunoZogy51,16 1,1984. 25. Janeway, C. A., Jr., Lemer, E. A., Mason, J. M., and Jones, B., Immunogenetics lo,48 1, 1980. 26. Gress, R. E., Wesley, M. N., and Hodes, R. J., J. Zmmunol. 127,1763, 1981. 27. Peck, A. B., Janeway, C. A., Jr., and Wigzell, H., Nature (London) 266,840, 1977. 28. Festenstein, H., Transpl. Rev. 15,62, 1973. 29. Festenstein, H., Transplantation 18,556, 1974. 30. Kyewski, B. A., Fathman, C. G., and Kaplan, H. S., Nature (London) 308,196,1984. 3 1. Kuhara, T., Hosono, M., and Fujiwara, M., Cell. Zmmunol. 57,377, 1981. 32. Bradley, S. M., Morrissey, P. J., Sharrow, S. O., and Singer, A., J. Exp. Med. 155, 1638, 1982. 33. Chervenak, R., Moorhead, J. W., and Cohen, J. J., J. Zmmunol. 134,695,1985. 34. Berumen, L., Halle-Pannenko, O., and Festenstein, H., Eur. J. Zmmunol. 13,292, 1983. 35. Berumen, L., Festenstein, H., and Halle-Pannenko, O., Immunogenetics 20,33,1984. 36. Click, R. E., Adelmann, A. M., and Azar, M. M., J. Zmmunol. 134,2948,1985. 37. Molnar-Kimber, K. L., and Sprent, J., Transplantation 31,376, 1981. 38. Janeway, C. A., Jr., and Katz, M. E., J. Zmmunol. 134,2064,1985. 39. Lutz, C., Glassbrook, A. L., and Fitch, F. W., Eur. J. Zmmunol. 11,726, 1981. 40. Ishizaka, S. T., and Stutman, O., Eur. J Zmmunol. 13,936, 1983.
IMMUNOLOGY
103,l l- 18 ( 1986)
Neonatal Tolerance Induction in the Thymus to MHC-Class II-Associated Antigens II. Significance
of MHC Antigens in Anti-MIS Tolerance
MASAMICHI HOSONO AND YOSHIMOTO KATSURA Department oJBacteriology and Serology, Chest Disease Research Institute, Kyoto University, Kyoto 606, Japan Received January 17,1986; acceptedJune 5,1986 Specificity of anti-Mlsa tolerance induced in BALB/c (H-2d, Mlsb) neonates was investigated by a pop&al lymph node (PLN)-swelling assay for the local graft-versus-host (GVH) reaction by injecting tolerant thymus cells into the footpads of several types of Fl hybrid mice. When thymus cells were obtained from l-week-old normal BALB/c, they evoked enlargement of PLNs of (BALB/c X DBA/Z)F 1(H-2d, Mlsb”) [CDF 1] recipients and of other hybrid recipients, heterozygous in Ml.+csdalleles, irrespective of the major histocompatibility complex (MHC) haplotypes. The same thymus cells did not causethe responsein MHC-heterozygous Fl hybrids when the hybrids were homozygous in Mlsb, identical with BALB/c mice. Therefore, the PLN response to Mls antigens, known to be closely associated with MHC-class II antigens, was not directed to the class II antigens themselves. This enabled us to examine the effects of MHC on tolerance induction to the Mls antigens. When BALB/c neonates were injected with CDFl bone marrow cells, complete tolerance to M~s’-H-~~antigens of CDFl cells was induced in the thymus, while responsivenessto Mls” antigens in the context of H-2’ and H-2b antigens, was not alEcted. This indicates MHC-restriction of neonatal tolerance to Mls antigens. Furthermore, when Mls and H-2-heterozygous (BALB/c X AKR)Fl (H-2W, Mlsb”) bone marrow cells served as the tolerogen, thymus cells of BALB/c neonates were also tolerized to Mls’-H-2’ antigens as well asto M~s*-H-~~antigens, which suggeststhe involvement of MHC, probably classII antigens of tolerance-inducing cells. 0 1986 Academic PITS, Inc.
INTRODUCTION Neonatal injection of major histocompatibility complex (MHC)-allogeneic and semiallogeneic hematopoietic cells induced unresponsiveness to the corresponding antigens asjudged at adulthood in in vivo (1,2) and in vitro (3-5) assaysystems. The results obtained by the tolerance induction method developed originally as a model of self tolerance, may not be extended to whole sets of self antigens, since recent reports suggest that the responses to allo-MHC antigens differ from those to nonMHC, minor antigens in terms of T cell subpopulations (6,7), physiology of antigenpresenting cells (8), and tolerance inducibility (9-l 1). Previously (12), we suggestedthat different mechanisms are involved in neonatal tolerance induction to allo-MHC antigens and non-MHC antigens and that immunological tolerance to the latter antigens to which the T-cell response is restricted to selfMHC antigens may provide a good model for self tolerance to non-MHC, conven11 0008-8749186$3.00 Copyrisht8 1986 by Academic
F’ms, Inc. AII rigbis of rcpraduction in any form resmed.
12
HOSONO AND KATSURA
tional antigens, as reported by others using different experimental systems ( 13-l 8). When we consider ontogenetic development of self tolerance, MHC-class II restriction of tolerance induction is of interest as the mechanism for negative selection of T cells committed to self antigens, since expression of Ia antigens is primarily localized in the thymus (19-24). Intravenous injection of Mls-semiallogeneic bone marrow cells readily induced neonatal tolerance in thymic T cells directing to the Mls antigens (12), to which Tcell activation responsesare closely associatedwith Ia antigens on the stimulator cells (25-27). In this respect, we examined whether the anti-Mullstolerance induction in newborn thymus is affected by the MHC haplotype of tolerance-inducing bone marrow cells. MATERIALS AND METHODS Most materials, methods, and experimental schedules employed in this study were as described in detail previously ( 12). Mice used here were BALB/c (H-2d, Mlsb), DBA/2 (H-2d, MIS’), BlO.BR (H-2k, Mlsb), C3H/He (H-2k, Mls”), and C57BL/6 (H-2b, Mlsb) purchased from the Shizuoka Agricultural Cooperative Association for Laboratory Animals (Shizuoka). AKR/J (H-2’, Ml?) and CBA/J (H-2k, Mlsd) mice were donated by Dr. S. Muramatsu, Faculty of Science, Kyoto University. Fl hybrids between these strains of mice were produced in our animal facility and used at 3 to 5 months of age. Tolerance was induced in BALB/c neonates (24 hr after birth) by intravenous injection of semiallogeneic bone marrow cells (2 X 10’ in 50 ~1). One week later, the mice were killed and their thymus cells were tested in vivo for their activity to cause the local graft-versus-host reaction (GVHR) by injecting thymus cells (2 X 10’ in 25 ~1)into the footpads of the Fl hybrids. Further 1 week later, popliteal lymph nodes (PLN) of each Fl hybrid recipient were removed and weighed. As negative controls, the medium was injected. The degree of PLN enlargement was expressed as lymph node (LN)-swelling index (LN index = PLN weight of cell-injected group/PLN weight of negative control group). Percentage of tolerance was calculated as follows: percentage tolerance = [ 1 - (mean PLN weight of tolerant group - mean PLN weight of negative control)/(mean PLN weight of non-tolerized group - mean PLN weight of negative control)] X 100, as previously described ( 12). RESULTS
Ontogenyof Thymus Cells Reactiveto Allo-MS and MHC-Class II Antigens Figure 1 shows the developmental profiles in ontogeny of anti-Mls and H-2 reactivities of thymus cells. Injection of thymus cells of BALB/c (H-2d, Mlsb) mice of various agesinto the footpads of (BALB/c X DBA/2)Fl (H-2d, Mlsb@) mice and (BALB/c X BlO.BR)Fl (H-2d/@,Mlsb) mice caused popliteal lymph node (PLN) enlargement as a result of a graft-versus-host reaction (GVHR) ( 12): the disparity in Mls and H-2 haplotypes in comparison with BALB/c mice is indicated in a circle. As shown in this figure, the activity to cause the anti-Ml!? GVHR was already appreciable at 24 hr of age, and reached plateau values by 1 week of age. In contrast, anti-H-2k activity was not detectable during the first 2 weeks of age. The delay in reaching the plateau value and low level of anti-allo-class II response, as compared with anti-Mls responsiveness,
MHC-ASSOCIATION
OF ANTI-Mls TOLERANCE
2
1
0
3
Weeks after
13
4
birth
FIG. I. Ontogeny of anti-Mls and Ia activity of BALB/c thymus cells. Thymus cells prepared from individual BALB/c (H-2d, Mlsb) mice of various agesas indicated on abscissa,were injected into the footpads of (BALB/c X DBA/2)Fl (H-2d, Mlsbl@)(0) and of (BALB/c X BlO.BR)Fl (H-2‘@, Mlsb) (0) mice for the local GVHR. Each symbol indicates the activity of each thymus. Horizontal striped bar represents + 1 SE of the mean of control groups ( 1.00 + 0.05 and 1.OOf 0.09 for anti-Mls’ and anti-I$ activity, respectively).
may reflect not only the different size of responding cell population but also the delayed maturation of anti-allo-class II activity. Unresponsiveness to allo-H-2 antigens of l-week-old BALB/c thymus cells was also observed for H-2b haplotypes of antigens as tested in (BALB/c X C5’7BL/6)Fl (H-2 dti Mlsb) hosts (Fig. 2). The lack of activity in l-week-old thymus cells was not attributable to the number of inoculated cells since fewer thymus cells showed no activity.
Demonstration of Anti-Mls Activity but not of Anti-allo-Class II Activity in I-WeekOld Thymus Anti-Mlsqd activity in 1-week-old BALB/c thymus cells were investigated by means of the local GVHR in various types of Fl hybrid recipients. These recipients were either homozygous or heterozygous for H-2d and/or Mlsb haplotypes, so that the involvement of host-versus-graft reaction could be excluded in the PLN-swelling assay.
4r
_I 0625
025
05
1.0
20
Na of thymus cells (x10’) FIG. 2. Cell dose effect on anti-Mls and H-2 GVHR. Various numbers of thymus cells of l-week-old BALB/c mice, as indicated on abscissa,were injected into (BALB/c X DBA/Z)Fl (H-2d, Ml@‘@)mice (0) and (BALB/c X C57BL/6)Fl (H-2@, Mlsb) mice (0) for assessinganti-Mls” and Iab activity, reqectively. Standard errors of the mean are represented by vertical bars, or omitted when they were smaller than the symbol marks. The horizontal striped bars represent f 1 SE of the mean of the control groups which received injections of medium (1.OO+ 0.12 and 1.oO+ 0.04 for anti-Mb’ and anti-Iab activity, respectively).
14
HOSONO AND RATSURA TABLE I Anti-Mls GVH Activity of l-Week-Old BALB/c Thymus Cells” Disparity in Group
Fl hybrid recipients (BALB/c X DBA/Z)F 1 (BALB/c X C57BL/6)Fl (DBA/Z X C57BL/6)Fl (BALB/c X BlO.BR)Fl (DBA/Z X BlO.BR)Fl (BALB/c X ARR)Fl (BALB/c X CBA/J)FI (BALB/c X C3H/He)Fl
H-2 -
b b k k k k k
Mls
LN index & SE (n)
a a a
3.23 +-0.19 (23) 1.15~0.05(11) 3.29 f 0.29 (7) 1.53+0.09(17) 3.22 + 0.37 (8) 2.78 f 0.11 (7) 2.56 + 0.24 (23) 2.04 + 0.16 (5)
Ii C
’ GVHR-inducing activity of 1-week-old, normal BALB/c thymus cells is expressed as LN index + SE in the PLN-swelling assayby injecting thymus cells into the footpads of Fl hybrid mice. Figures in parenthesesindicate the number of PLNs tested.
As summarized in Table 1, the newborn thymus cells caused PLN enlargement when recipients were heterozygous in Mls”, Mls”, and Mlsd, irrespective of MHC haplotypes (Groups 1, 3, and 5-8): stronger responses to Mlsasdantigens than to Mls” antigens was in agreement with the in vitro results in the mixed lymphocytes reaction (28, 29). On the other hand, they had no effect on the Mls-identical, MHC-heterozygous recipients (Groups 2 and 4). Thus, l-week-old thymus cells appear to be responsive to allo-Mls antigens but not to allo-class II antigens.
MHC Restriction ofAnti-Mls ToleranceInduction As shown in the top groups of experiments A and B of Fig. 3, the thymus cells became tolerant (80-90s) to M~s*-H-~~ antigens and showed only the background level response of medium controls. The same samples of thymus cells, on the other hand, evoked stron GVHR in (BALB/c X AKR)Fl (H-2d/@,Mlsb@) and (C57BL/6 X DBA/2)F 1 (H-2 8b’d Mlsb@) hosts, both of which express Mls” antigens and allogeneic H-2 antigens, and in (BALB/c X CBA/J)Fl (H-2d’@, Mlsb@) hosts, expressing M~s~-H-~~““antigens. These results were not attributed to reactivity of the thymus cells to allogeneic H-2 antigens themselves, since they had no effect on (BALB/c X BlO.BR)Fl (H-2d@, Mlsb) or (BALB/c X C57BL/6)Fl (H-2d’@,Mlsb) hosts.
MHC Haplotypes of Tolerance-InducingCells in Anti-k% Tolerance We examined whether the MHC haplotype of tolerance-inducing cell donors is responsible for the MHC restriction in anti-Mls neonatal tolerance, using MHC-heterozygous (BALB/c X AKR)F 1 (H-2d’@,Mlsb@) bone marrow cells as tolerogens. As shown in Table 2, the percentage of tolerance for the corresponding M~s~-H-~~~test antigens was higher than that for M~s”-H-~~antigens of (BALB/c X DBA/2)Fl mice (Groups 1 and 2), suggesting that potential responsiveness to M~s”-H-~~ antigens, in addition to M~s~-H-~~antigens, is also suppressed.Such hyporesponsiveness is again specific to the Mls antigens in association with MHC haplotypes of the tolerance-
MHC-ASSOCIATION Hybrid recipients (H-2 1 MIS) W A (BALBk xDbv2P
OF ANTI-Mb
15
LN index N
.b@)
T
(BA!eB/c x AKR)R I dC3.W)
7
M&cx~BA)FI
:
‘BAI.~
b” FlO BR)Fl
;
‘“;“;,B$
; ~57WFt
;
(d
TOLERANCE
FIG. 3. Specificity of neonatally induced tolerance to the Mls antigen. BALB/c (H-2d, Mlsb) neonates were injected with bone marrow cells of (BALB/c X DBA/2)Fl (H-2d, Mlsb’@)mice. Thymus cells of the tolerized BALB/c mice were injected into the footpads of Fl hybrids homozygous or heterozygous in H-2d and Mlsb as indicated (T, w). The same numbers of thymus cells of age-matched, normal mice were used as positive controls (N, 0). One week after the footpad injection, the Fl mice were killed and their popliteal lymph nodes were weighed for the GVHR. The horizontal column with bar represents the mean LN index with + 1 SE and the vertical striped bar represents + 1 SE of the mean of negative control group receiving injections of medium. In experiment B, the bone marrow cells used as tolerogens, were pretreated with anti-Thy. 1 monoclonal antibody plus complement to remove T cells, if any, before injection.
inducing cells, the GVHR-inducibility (Group 3).
to M~s”-H-~~ antigens being not affected
Antigen Specificity ofAnti-Mls” Tolerancein the Thymus BALB/c neonates again were tolerized with (BALB/c X AKR)Fl bone marrow cells, and the antigen specificity of anti-Mls” tolerance in the thymus was tested. Table 3 shows the results. The degree of tolerance to M~s~-H-~~* antigens and M~s”-H-~~ antigens were 75 and 5 l%, respectively (Groups 1 and 2), confirming the previous experimental results. The same sample of thymus cells showed 49% cross tolerance to M~s~-H-~~~antigens of (BALB/c X CBA/J)Fl mice (Group 3), and normal activity to M~s”-H-~~~ antigens of (BALB/c X C3H/He)Fl mice (Group 4). DISCUSSION To find a clue for understanding the mechanisms of self tolerance in normally developing thymus cells contributing in response to MHC-class II-associated antigens, the Mls antigens seemed to be appropriate antigens, because of their strong immunogenicity in primary response in a fashion of self-class II restriction (25-27) not only for peripheral mature T cells but also for thymus cells ( 11, 12). Furthermore, neonatal tolerance to this antigen is relatively easily induced in thymus and spleen T cells within 1 week and continues for at least 4 months after birth. As we showed, the induction of neonatal tolerance to Mls antigens seemsto be associated with MHC-
16
HOSONO AND KATSURA TABLE
2
Restriction of Anti-Ml9 Tolerance Induction to the MHC Haplotype of Tolerance-Inducing Bone Marrow Cells” stateof Fl
hybrid recipients[H-2,
1 (BALB/c
X AKR)FI
[d/B,
Mls] b/@]
2 (BALB/c
X DBA/2)Fl
[d, b/@]
3 (DBA/Z
X C57BL/6)Fl
[d/B,
4 (BALB/c
X C57BL/6)Fl
[d/Q,
a/b] b]
BALB/c mice
LN index + SE(n)
Nor. Tol. Nor. Tol. Nor. Tol. Nor. Tol.
2.75 + 0.15 (4) 1.42+0.18(4) 3.56 + 0.20 (5) 2.28 + 0.16 (5) 3.77 f 0.56 (3) 3.15*0.16(3) 1.15 kO.lO(4) 1.53 f 0.04 (3)
P -
96 To1e.ranc-e -
0.02 -
77 -
0.01 -
50 -
0.70 -
Nontol.’ -
0.06
Nontol.
a BALB/c [H-2d, Mlsb] neonates received bone marrow cells of (BALB/c X AKR)Fl [H-2@, MlsWO] mice. One week later, thymus cells of the cell-injected BALB/c mice (Tol.) were routinely tested for GVHR inducibility in various types of Fl hybrid mice.Thymescellsof uninjectedlittermatesor age-matchedmicewereusedaspositivecontrols(Nor.). b Nontol. = nontolerant (P > 0.05).
haplotypes of the tolerance-inducing bone marrow cells, confirming the findings that tolerance to non-MHC antigens is restricted to self-MHC antigens (13-l 8). Together with the fact that Ia antigens appear first in the thymus (19-24), MHC restriction of class II-reactive T-cell tolerance predicts that the thymus is a site of clonal elimination of the classII-reactive T cells committed to self antigens, especially at an early stage of development when there are few Ia+ cells in periphery, and a site of Ia-expression may be closely related to the site where tolerance induction of the class II-reactive T cells is initiated. In this sense,tolerance to peripheral, non-MHC antigens would occur after their entrance into the thymus and in association with Ia antigens of thymic non-lymphoid cells (30). On the other hand, it is reasonable to assume that the extrathymic presence of Ia antigen-bearing cells in the periphery, in unphysiological or experimental conditions, aEectsT-cell repertoire generation, if Tcell receptors are prethymically expressed,as suggestedelsewhere ( 11,3 l-33). TABLE 3 Antigen Specificity of Anti-Ml9 Tolerance in the Thymus” stateof BALBjc Fl
hybrid recipients[H-2, Mls]
1 (BALB/cXAKR)Fl 2 (BALB/c 3
X DBA/Z)Fl
[d/@,b/@] [d, b/ @]
(BAWc X CWJW Id/ @ , b/ @ 1
4 (BALB/c
X C3H/He)Fl
[d/ @ , b/Q]
mice
LN index k
SE (n)
P
w Tolerance -
Nor.
4.92 f 0.82 (5)
-
Tol. Nor.
2.00 f 0.36 (4) 3.83 f 0.21 (5)
0.01 -
75 -
Nor.
Tol.
2.36 f O.ll(6) 2.77 + 0.1 I (5)
0.01 -
51 -
Tol. Nor. Tol.
1.91 f 0.07 (4) 2.04 f 0.16 (5) 1.9OiO.11 (6)
0.01 0.66
49 Nontol.b
’ Experimental system and schedule were the same as those employed in Table 2, except for types ofF1 hybrid recipients used for GVHR. b Nontol. = nontolerant (P > 0.05).
MHC-ASSOCIATION
OF ANTI-Mls TOLERANCE
17
However, this may not be the casefor the Mls antigens, since Morrissey et al. (11) showed that, in radiation chimera experiments, tolerance to the antigens was only induced intrathymically. Therefore, in our model of neonatal tolerance, entrance into the thymus of donor stem cell-originated non-lymphocytes, Ia positive, must be a requisite for the negative selection process. Such dependence of anti-Mls-tolerance induction on Ia-bearing non-lymphocytes in developing thymus may provide a useful model for the analysis of biological significance of intrathymic expression of Ia antigens in early stageof ontogeny, since the degree of Mls-specific tolerance depends on the number of tolerance-inducing cells injected and on MHC (Mls)-gene dosage, MHC (Mls)-homozygous tolerogen being twice as tolerogenic as MHC (Mls)-heterozygous tolerogen ( 12). Although we have tentatively referred to the Mls antigen as a non-MHC conventional antigen, as has been indicated by Berumen et al. (34,35) and Click et al. (36), the identity and structure of the Mls antigen is controversial, since the molecular nature of the antigen has not yet been defined. Janeway et al. (38) recently proposed that the Mls-gene functions in stabilizing T-cell-Ia antigen interaction rather than in producing a particular immunogenic component. According to Janeway’s model, Mls-gene product(s) on Ia-bearing, antigen-presenting cells (APC) stabilize the T-cell-APC interaction, so that preferential stimulation of low affinity self-class II-reactive T cells would occur, as in autologous mixed lymphocyte reaction. This would explain the high frequency of Mls-reactive T cells (39, 40) and probably as a result, the early appearance of anti-Mls reactivity in the thymus. As mentioned above, neonatal tolerance to “Mls antigens” is closely associated with MHC-haplotypes of tolerogen and tolerance to M~s”-H-~~‘~ antigens showed also reduced responsiveness to M~s~-H-~~~antigens. Thus, the model could explain most of our experimental observations. However, it should be noted that in preliminary experiments with the local HVG assaysystem (BALB/c X CBA/J)Fl (H2d/k, Mlsbld) mice responded to M~s”-H-~~‘~ antigens of (DBA/2 X BlO.BR)Fl (H-2dF, Ml@‘) mice, while no response was observed in the reciprocal donor-recipient combinations, suggestingthat T-cell population directing to the Mlsd antigens are not identical to those to the Mls” antigens, and that tolerance to M~s”-H-~~~ antigens did not affect the responsivenessto M~s”-H-~~~antigens, as reported here, suggesting that the Mls”-gene function differs from that of Mls”-gene. These data are in agreement with the concept of polymorphism in the Mls-gene function. Studies on the specificity of tolerance to MlsdYcantigens, in addition to Mls” antigens, are in progress. ACKNOWLEDGMENT The work was supported by a grant for the Shimizu Foundation Research.
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18
HOSONO AND KATSURA
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