CD30 is a member of the TNF receptor superfamily that has been implicated in negative selection and some forms of peripheral tolerance. A previous study of CD30−/− mice in a class I-restricted H-Y TCR-transgenic mouse model showed that CD30 is essential for removal of autoreactive thymocytes. During the course of the studies of CD30 in the class II-restricted TCR-transgenic mice, we found that the absence of CD30 has no effect on negative selection. Surprisingly, we also found that the CD30 mutation does not perturb apoptosis of the autoreactive thymocytes in the class I-restricted H-Y TCR-transgenic model. The minimal role of CD30 in negative selection and other recent data are discussed.

Apoptosis plays a prominent role in the maintenance of self-tolerance in the immune system. Autoreactive T cells are removed through apoptosis during development in the thymus (negative selection) and after maturation in the peripheral immune organs (1, 2). Members of the TNF receptor superfamily play a prominent role in peripheral tolerance (3, 4, 5, 6). Mutation in the Fas gene in lpr/lpr mice, for example, leads to development of autoimmune disease (7), characterized by uncontrolled lymphoproliferation, splenomegaly, and the presence of anti-dsDNA Abs (8). TNF receptor type I is also implicated in peripheral tolerance, as its absence exacerbates the lpr/lpr mouse phenotype (9). Both Fas and TNF receptor type I encode cytoplasmic tails containing a death domain motif (3). Upon ligand engagement, the death domain mediates interaction with the adapter molecule FADD/Mort1, which recruits the downstream caspases, leading to apoptosis (5, 10, 11). Fas has also been implicated in some aspects of thymic apoptosis (12) but negative selection is largely intact in lpr/lpr mice. In contrast, mice lacking another member of the TNF receptor family, CD30, were reported to be defective in negative selection (13).

CD30 is a molecule that was first identified as a cell surface marker of Reed-Sternberg and Hodgkin’s cells of Hodgkin’s disease patients. CD30 is also found on a variety of other non-Hodgkin’s lymphomas, on virally transformed T and B cell blasts, as well as on the surface of HIV-infected lymphocytes (14, 15). CD30 is normally expressed on activated T and B cells and therefore appears to be associated with an activated phenotype. Interestingly, in contrast to the other TNF receptor family members that can clearly deliver apoptotic signals, CD30 does not contain a death domain in its cytoplasmic tail. CD30 has been shown to associate with TNFR-associated factor (TRAF)3 1 and TRAF2, which signal activation of NF-κB (16, 17, 18, 19). However, no apoptosis-specific signaling molecules have been identified for the CD30 pathway.

Another molecule known to be involved in thymic cell death is the transcription factor Nur77, whose expression is induced in response to TCR stimulation (20, 21). Dominant negative Nur77-transgenic mice exhibit partial defects of negative selection. In contrast, constitutive expression of Nur77 (Nur77-FL) leads to massive thymic apoptosis (22). During our efforts to further define the downstream targets for Nur77, we found that CD30 levels were elevated on thymocytes from Nur77FL mice compared with the wild-type littermates. Subsequent analysis of Nur77-FL mice in CD30−/− background, however, revealed that CD30 does not play a direct role in Nur77-mediated apoptosis in the thymus (22). To confirm the role of CD30 in negative selection, we examined the effect of CD30 mutation on apoptosis mediated by alloreactivity against I-As in transgenic mice that express the cytochrome c/I-E-specific TCR (23). We were surprised to find that the CD30 deficiency failed to rescue thymocytes from negative selection in these AND mice. We have since obtained similar data with the class I-restricted H-Y TCR-transgenic mice (24). Apoptosis of male Ag-specific H-Y TCR-transgenic T cells occurs normally in the CD30−/− background. We conclude that in contrast to a previous report (13), CD30 does not play a role in negative selection.

C57BL/6 and AND mice were obtained from The Jackson Laboratory (Bar Harbor, ME). AND mice were crossed to SJL mice (H-2s). Both H-Y(H-2b) and AND × SJL (H-2b/s) mice were crossed with CD30−/− mice (a generous gift from Pamela Ohashi and Tak Mak, Ontario Cancer Institute, Toronto, Ontario, Canada). Mice were analyzed at 5–10 wk of age.

PCR analysis was used to genotype mice from the CD30−/− × TCR-transgenic mice. CD30 primers used were as previously described (13) using CD30g-(common) primer, 5′-CAACCCTGGCTGAGTTACTC, CD30 wild-type primer, 5′-AGCGGCAGGTTCTTCAGGTA, and tv30neo primer 5′-TATCAGGACATAGCGTTGGCTACC. For typing H-Y-transgenic mice, the H-Y PCR primers to the TCR β-chain are HYF 5′-AACAGGAGGAAAGGTGACATTGAG and HYB 5′-GGACAAAAACTGGCTCTGGCTATC. For AND TCR mice, they were genotyped using primers for TCR gene Vα11 (5′-GCTCAGCTAAGTTCCTATTA) and Jα84 (5′-TGGAATCCATAGAAAGAGAC). The MHC backgrounds were typed using primers specific for H-2b (5′-CCATGGTTTTTGGAATACTG and 5′-CCGCACAAGGAATTTATCCG) and H-2s (5′-CCATGGTTTTTGGAATATTC and 5′-CGGCACAAGGAATTTATCCA).

Thymi from CD30−/− and C57BL/6 mice were removed and homogenized using a VIRTISHEAR homogenizer (Virtis, Gardiner, NY). Total RNA was isolated using the RNase-All method (25). Total RNA was run on a 1.2% formaldehyde gel, 1× MOPS buffer and blotted overnight onto nylon filter. Hybridization was done using the ExpressHyb hybridization solution (Clontech, Palo Alto, CA). Murine CD30 cDNA (a generous gift from Eckhard Podack, Miami University, Miami, FL) was labeled with 32P by a random priming method and used as a probe for the Northern blot.

Thymocytes were isolated from mice at 5–10 wk of age and teased apart in tissue culture media with glutamine (Life Technologies, Grand Island, NY). Cells were stained with mAbs to CD4, CD8 α, αβTCR (Caltag, South San Francisco, CA), Vα11 (PharMingen, San Diego, CA), and clonotypic anti-H-Y Ab (T3.70) (a generous gift from James Allison, University of California, Berkeley). Cells were stained in 1× PBS (0.2 g/L KCl, 0.2 g/L KH2PO4, 8.0 g/L NaCl, 2.16 g/L Na2HPO4.7H2O), 2% FCS, and 0.1% sodium azide on ice for 20 min and then analyzed using a Coulter flow cytometer (Coulter Pharmaceutical, Palo Alto, CA).

Disruption of the CD30 gene has previously been shown to rescue the deletion of self-reactive thymocytes in an H-Y TCR-transgenic model but not in superantigen-mediated negative selection model (13). Our experiments with the CD30−/− Nur77FL-transgenic mice have shown that CD30 is not involved in thymic apoptosis caused by Nur77 overexpression (22). In an effort to characterize CD30 further, we undertook a brief study of the CD30−/− mice. CD30 knockout mice were previously generated by replacement of the second to last exon of the CD30 gene with the neomycin-resistant gene (13). To rule out the possibility that there may be residual activity from a truncated or aberrantly expressed CD30 molecule, we isolated total thymic RNA from CD30−/− and wild-type C57BL/6 mice. We then probed the RNA with the murine CD30 cDNA in a Northern blot analysis. We found that CD30 transcript was absent in the thymus of CD30−/− mice (Fig. 1 A). In contrast, CD30 expression was visible in the C57BL/6 thymus as had been shown previously (26). These results confirm the lack of CD30 transcript in CD30−/− mice.

FIGURE 1.

Northern blot and genotype analysis of CD30-deficient mice. A, Northern blot analysis of total RNA from wild-type (C57BL/6) and CD30−/− thymocytes using CD30 cDNA as a probe. Ethidium bromide gel photograph of the corresponding RNA is shown below. B, Genotype analysis of the wild-type, CD30+/−, or CD30−/− mice. Two PCR are used to distinguish the CD30 genotypes; k/o, the CD30 knockout PCR product; wt, the wild-type CD30 allele.

FIGURE 1.

Northern blot and genotype analysis of CD30-deficient mice. A, Northern blot analysis of total RNA from wild-type (C57BL/6) and CD30−/− thymocytes using CD30 cDNA as a probe. Ethidium bromide gel photograph of the corresponding RNA is shown below. B, Genotype analysis of the wild-type, CD30+/−, or CD30−/− mice. Two PCR are used to distinguish the CD30 genotypes; k/o, the CD30 knockout PCR product; wt, the wild-type CD30 allele.

Close modal

We mated CD30−/− mice to AND TCR-transgenic animals, a well-studied model of thymic selection. As a control, we also crossed CD30−/− mice to the H-Y TCR-transgenic mice. The AND TCR is specific for pigeon cytochrome c and Ek. In transgenic animals, transgene-expressing T cells are positively selected on Ab molecule whereas negative selection occurs in the presence of As (23). Negative selection can also be initiated by injection of pigeon cytochrome c peptide with mice in the H-2k/b background. The alloreactive negative selection, however, is milder than peptide-initiated apoptosis and does not involve killing by TNF secreted by the stimulated transgenic peripheral T cells (27). The genotypes for CD30 TCR-transgenic mice and their MHC haplotypes were determined by PCR analysis (Fig. 1,B and data not shown). Analysis of these mice show that transgenic T cells are positively selected in the H-2b background, with skewing toward the CD4+CD8 lineage as shown before (23). In the CD30 heterozygous, H-2s background, negative selection takes place as evidenced by the absence of positive selection and severalfold drop of thymocyte cell number (Fig. 2). In CD30−/− H-2s animals, however, thymocyte cell numbers and the FACS profiles remained similar to those in the heterozygous littermates (30 millions, n = 6 vs 37 millions thymocytes, n = 5). No increase in the representation or the number of CD4+CD8+ thymocytes was seen, as would be predicted if CD30 was involved in negative selection (Fig. 2). Cells were also stained with AND TCR-specific Ab (anti-Vα11) and again there were no differences in the staining profiles or absolute cell numbers among CD30+/−, AND H-2s and CD30−/−, and AND H-2s mice (data not shown).

FIGURE 2.

Lack of CD30 has no effect on class II-restricted negative selection. Representative plots of CD4 and CD8 expression on thymocytes from AND-transgenic mice in CD30 heterozygous (+/−) or homozygous (−/−) mutant background. The MHC haplotypes of the mice analyzed and their average total thymocyte numbers are indicated.

FIGURE 2.

Lack of CD30 has no effect on class II-restricted negative selection. Representative plots of CD4 and CD8 expression on thymocytes from AND-transgenic mice in CD30 heterozygous (+/−) or homozygous (−/−) mutant background. The MHC haplotypes of the mice analyzed and their average total thymocyte numbers are indicated.

Close modal

Originally serving as a control, we also bred CD30−/− mice with the H-Y TCR-transgenic line. The H-Y TCR is specific for the H-Y Ag presented in the context of Db molecule. In female mice, H-Y TCR-bearing T cells are positively selected on H-2b molecules whereas in male mice, transgenic T cells are negatively selected due to the presence of the H-Y Ag. To exclude T cells expressing any endogenous TCR-α, we gated the FACS profiles on T3.70+ cells (T3.70 is a clonotypic Ab specific for the H-Y TCR). Thymocytes were stained with anti-CD4 and anti-CD8 Abs. As shown in Fig. 3, we were surprised to see no difference in the thymocyte profiles of male CD30+/− HY vs CD30−/− HY mice. As expected, the cell number of male H-Y CD30+/− thymocytes is reduced dramatically compared with the nontransgenic littermate or female H-Y (either CD30+/− or CD30−/−) mice (Fig. 3). However, there was no increase in the representation of the double-positive (CD4+CD8+) thymocyte population or in the absolute cell number of thymocytes from male H-Y TCR CD30−/− mice compared with the CD30+/− counterpart. At least six different littermates were analyzed with similar results. Combined with the AND data above, these unexpected results have led us to conclude that apoptosis by negative selection proceeds normally in CD30−/− mice.

FIGURE 3.

Negative selection mediated by the H-Y male Ag proceeds normally in the absence of CD30. Representative plots of CD4 and CD8 expression on gated T3.70+ thymocytes from H-Y male and female transgenic mice in CD30 heterozygous (+/−) or homozygous (−/−) mutant background.

FIGURE 3.

Negative selection mediated by the H-Y male Ag proceeds normally in the absence of CD30. Representative plots of CD4 and CD8 expression on gated T3.70+ thymocytes from H-Y male and female transgenic mice in CD30 heterozygous (+/−) or homozygous (−/−) mutant background.

Close modal

CD30 was initially identified as a marker for Hodgkin’s disease. Further studies of CD30 have implicated a role for CD30 in negative selection and peripheral tolerance of CD8+ cells (4, 13, 15). However, our study of two different TCR-transgenic systems, AND and H-Y, points to a conclusion that CD30 plays little or no role in the removal of autoreactive cells in the thymus. The question arises then, as to how to reconcile the results obtained with the H-Y TCR-transgenic model in our study with that of the previously published results by Amakawa et al. (13). One explanation may lie in the differences in the mouse strains used for the experiments. We have used CD30 mutant mice that have been crossed repeatedly to C57BL/6. In contrast, Amakawa et al. (13) might have used strains that were bred only for a few generations to C57BL/6. Another explanation may lie in the conditions under which the mice are housed. Nonetheless, the AND and H-Y TCR-transgenic mice show no defects in negative selection in the absence of CD30. These data argue for a minor role for CD30 in thymic apoptosis.

Recently, overexpressing CD30 in transgenic mice was shown to enhance negative selection but only in the presence of TCR signals (28). The authors conclude that CD30 may act as a costimulatory molecule for negative selection. Using Abs specific for cell surface molecules, however, Kishimoto and Sprent (29) found that anti-CD28 was the only Ab that could provide costimulatory activity for TCR-dependent apoptosis of CD4+CD8+ thymocytes in vitro. Negative results were observed for Ab specific for CD30 (29). Furthermore, CD30 expression is confined to the thymic medulla (14, 30). This is incompatible with the purported role of CD30 in CD4+CD8+ thymocytes, which reside in the cortex. Studies of CD30 signal transduction have revealed TRAF1 and TRAF2 as two signaling proteins that bind to CD30 (16, 17, 18, 19). Upon ligand engagement, these TRAF molecules mediate NF-κB activation, which has an antiapoptotic activity. These data, along with our TCR-transgenic experiments, argue for an alternative role of CD30. Further detailed studies of CD30−/− mice are needed to re-examine the role of CD30 in the immune system.

We thank Drs. P. Ohashi and T. Mak for the CD30 mutant mice, Dr. E. Podack for the CD30 cDNA clone, and Dr. Sue Sohn for critical reading of this manuscript.

1

This work was supported by the National Institute of Health Grant CA66236 (to A.W.). A.W. is a National Science Foundation Presidential Faculty Fellow.

3

Abbreviation used in this paper: TRAF, TNFR-associated factor.

1
Nossal, G. J. V..
1994
. Negative selection of lymphocytes.
Cell
76
:
229
2
Amsen, D., A. M. Kruisbeek.
1998
. Thymocyte selection: not by TCR alone.
Immunol. Rev.
165
:
209
3
Nagata, S., P. Golstein.
1995
. The Fas death factor.
Science
267
:
1449
4
Kurts, C., F. R. Carbone, M. F. Krummel, K. M. Koch, J. Miller, W. R. Heath.
1999
. Signalling through CD30 protects against autoimmune diabetes mediated by CD8 T cells.
Nature
398
:
341
5
Wallach, D., E. E. Varfolomeev, N. L. Malinin, Y. V. Goltsev, A. V. Kovalenko, M. P. Boldin.
1999
. Tumor necrosis factor receptor and Fas signaling mechanisms.
Annu. Rev. Immunol.
17
:
331
6
Lenardo, M., F. K.-M. Chan, F. Hornung, H. McFarland, R. Siegel, J. Wang, L. Zheng.
1999
. Mature T lymphocyte apoptosis-immune regulation in a dynamic and unpredictable antigenic environment.
Annu. Rev. Immunol.
17
:
221
7
Watanabe, F. R., C. I. Brannan, N. G. Copeland, N. A. Jenkins, S. Nagata.
1992
. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis.
Nature
356
:
314
8
Cohen, P. L., R. A. Eisenberg.
1991
. Lpr and gld: single gene models of systemic autoimmunity and lymphoproliferative disease.
Annu. Rev. Immunol.
9
:
243
9
Zhou, T., C. K. R. Edwards, P. Yang, Z. Wang, H. Bluethmann, J. D. Mountz.
1996
. Greatly accelerated lymphadenopathy and autoimmune disease in lpr mice lacking tumor necrosis factor receptor I.
J. Immunol.
156
:
2661
10
Ashkenazi, A., V. M. Dixit.
1998
. Death receptors: signaling and modulation.
Science
281
:
1305
11
Scaffidi, C., S. Kirchhoff, P. H. Krammer, M. E. Peter.
1999
. Apoptosis signaling in lymphocytes.
Curr. Opin. Immunol.
11
:
277
12
Kishimoto, H., C. D. Surh, J. Sprent.
1998
. A role for Fas in negative selection of thymocytes in vivo.
J. Exp. Med.
187
:
1427
13
Amakawa, R., A. Hakem, T. M. Kundig, T. Matsuyama, J. J. L. Simard, E. Timms, A. Wakeman, H.-W. Mittruecker, H. Griesser, H. Takimoto, et al
1996
. Impaired negative selection of T cells in Hodgkin’s disease antigen CD30-deficient mice.
Cell
84
:
551
14
Horie, R., T. Watanabe.
1998
. CD30: expression and function in health and disease.
Semin. Immunol.
10
:
457
15
Heath, W. R., C. Kurts, I. Caminschi, F. R. Carbone, J. F. Miller.
1999
. CD30 prevents T-cell responses to non-lymphoid tissues.
Immunol. Rev.
169
:
23
16
Gedrich, R. W., M. C. Gilfillan, C. S. Duckett, J. L. Van Dongen, C. B. Thompson.
1996
. CD30 contains two binding sites with different specificities for members of the tumor necrosis factor receptor-associated factor family of signal transducing proteins.
J. Biol. Chem.
271
:
12852
17
Lee, S. Y., S. Y. Lee, G. Kandala, M.-L. Liou, H.-C. Liou.
1996
. CD30/TNF receptor-associated factor interaction: NF-κB activation and binding specificity.
Proc. Natl. Acad. Sci. USA
93
:
9699
18
Duckett, C. S., C. B. Thompson.
1997
. CD30-dependent degradation of TRAF2: implications for negative regulation of TRAF signaling and the control of cell survival.
Genes Dev.
11
:
2810
19
Duckett, C. S., R. W. Gedrich, M. C. Gilfillan, C. B. Thompson.
1997
. Induction of nuclear factor κB by the CD30 receptor is mediated by TRAF1 and TRAF2.
Mol. Cell. Biol.
17
:
1535
20
Calnan, B., S. Szychowski, F. K. M. Chan, D. Cado, A. Winoto.
1995
. A role of the orphan steroid receptor Nur77 in antigen-induced negative selection.
Immunity
3
:
273
21
Zhou, T., J. Cheng, P. Yang, Z. Wang, C. Liu, X. Su, H. Bluethmann, J. D. Mountz.
1996
. Inhibition of Nur77/Nurr1 leads to inefficient clonal deletion of self-reactive T cells.
J. Exp. Med.
183
:
1879
22
Zhang, J., A. DeYoung, H. G. Kasler, N. H. Kabra, A. A. Kuang, G. Diehl, S. J. Sohn, C. Bishop, A. Winoto.
1999
. Receptor-mediated apoptosis in T lymphocytes.
Cold Spring Harbor Symp. Quant. Biol.
64
:
363
23
Vasquez, N., J. Kaye, S. M. Hedrick.
1992
. In vivo and in vitro clonal deletion of double-positive thymocytes.
J. Exp. Med.
175
:
1307
24
von Boehmer, H..
1990
. Developmental biology of T cells in T cell-receptor transgenic mice.
Annu. Rev. Immunol.
8
:
531
25
Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, K. Struhl.
1990
.
Current Protocols in Molecular Biology
Wiley, New York.
26
Bowen, M. A., R. K. Lee, G. Miragliotta, S. Y. Nam, E. R. Podack.
1996
. Structure and expression of murine CD30 and its role in cytokine production.
J. Immunol.
156
:
442
27
Martin, S., M. J. Bevan.
1997
. Antigen-specific and nonspecific deletion of immature cortical thmocytes cause by antigen injection.
Eur. J. Immunol.
27
:
2726
28
Chiarle, R., A. Podda, G. Prolla, E. R. Podack, G. J. Thorbecke, G. Inghirami.
1999
. CD30 overexpression enhances negative selection in the thymus and mediates programmed cell death via a Bcl-2-sensitive pathway.
J. Immunol.
163
:
194
29
Kishimoto, H., J. Sprent.
1999
. Several different cell surface molecules control negative selection of medullary thymocytes.
J. Exp. Med.
190
:
65
30
Romagnani, P., F. Annunziato, R. Manetti, C. Mavilia, L. Lasagni, C. Manuelli, G. B. Vannelli, V. Vanini, E. Maggi, C. Pupilli, S. Romagnani.
1998
. High CD30 ligand expression by epithelial cells and Hassal’s corpuscles in the medulla of human thymus.
Blood
91
:
3323