Appearance of extrathymic early differentiated CD4-CD8- T cells with T cell receptor gamma/delta or alpha/beta after thymus grafting to nude mice: influence of thymus on extrathymic T cell differentiation.

Fetal thymus grafting into athymic nude mice has been used as an experimental model of T cell development. To understand the early events of T cell development, we have examined the sequence of appearance of T cell subsets in lymph nodes (LN) of BALB/c nu/nu mice after grafting with syngeneic fetal thymus. T cells expressing T cell receptor (TCR) alpha/beta or gamma/delta increased in LN from 1 week after grafting, although no host-derived CD3+ T cells were detected in the grafted thymus and no donor thymus-derived T cells were detected in the LN. The early appearing T cells of both TCR alpha/beta and TCR gamma/delta showed a CD4-CD8- phenotype. V region usage analysis of the early appearing TCR alpha/beta T cells revealed that they contained cells bearing V beta 3 or V beta 11, which are potentially reactive to self-superantigen Mls-2a or Dvb11, respectively, and are deleted in the course of T cell development in the thymus of euthymic BALB/c mice. The early appearing T cells showed neither mixed lymphocyte reaction nor cytotoxic T cell activity against allogeneic cells. In contrast, lymphokine-activated killer cells from early appearing T cells, which contained high percentages of TCR gamma/delta T cells, exhibited higher cytotoxic activity against P815 mastocytoma than those from euthymic mice or untreated nude mice. All these results suggest that the early appearing T cells are developed extrathymically. We propose that the thymus may induce extrathymic T cell development without direct cell-to-cell interaction. It seems likely that the extrathymically developed T cells, especially TCR gamma/delta T cells, induced by the thymus have some role in the defense mechanism in the absence of conventional thymus-derived T cells.

[Changes in bone and lipid metabolisms following oophorectomy and effects of estrogen replacement therapy].

The bone and lipid metabolisms in Japanese oophorectomized (OVX) women and effects of estrogen replacement therapy (ERT) were investigated. Three hundred and two OVX women (mean age of 41.6) and 164 premenopausal women (mean age of 42.1) were examined. BMD of the lumbar spine, serum Ca, P, Al-p, total cholesterol (T-C), HDL-C and LDL-C were measured every three months. BMD within 2 years after OVX was not significantly decreased. The mean BMD between 2 and 5 years or over 10 years after OVX was 0.83 (p < 0.01) and 0.69 (p < 0.001), respectively. Serum Ca and P were increased for 2 years and Al-p was increased for 10 years after OVX. To investigate the effect of ERT on BMD, OVX women were divided into 3 groups: ERT, VD3 and no treatment groups. After 12 months of treatment, the mean % BMD in each group was 102.9, 100.6 and 97.8, respectively. T-C or LDL-C was gradually increased in 10 years after OVX. After 15 months of ERT, the mean percentages HDL-C and LDL-C in hyperlipidemic women (LDL-C > or = 150) were 110.5 (p < 0.05) and 75.2 (p < 0.001). ERT may be requisite for OVX women and effective in preventing increased bone turnover and hyperlipidemism following OVX.

Failure of T cell receptor V beta negative selection in murine intestinal intra-epithelial lymphocytes.

The intestinal intra-epithelial lymphocytes (IEL) are divided into several subsets on the basis of expression of T cell receptor (TCR) alpha beta and gamma delta, intensity of Thy-1 expression and expression of Lyt-3 chain. To investigate the differentiation pathway of the IEL, we examined the repertoire of V beta segments of T cells in the IEL in BALB/c (H-2d, MIs-1b2a) or AKR/J (H-2k, MIs-1a2b) mice. Among freshly isolated IEL, an appreciable number of T cells bearing V beta 3 or V beta 11, which recognize MIs-2a- or MHC IE-encoded molecules respectively, were detected in BALB/c mice. Similarly, in AKR/J mice, IEL contained appreciable levels of V beta 6-bearing T cells. V beta 3- or V beta 11-bearing T cells in the IEL in BALB/c mice increased to a significant level when incubated with staphylococcal enterotoxin A which specifically stimulates V beta 3- and V beta 11-bearing T cells. Most of IEL without clonal deletion expressed Lyt-2 but not Lyt-3 antigens. Such T cells were hardly detected in other organs, including liver. Our results indicate that TCR alpha beta-bearing intestinal IEL that have not undergone negative selection may have differentiated outside the thymus, presumably at a local site of the intestine and can respond normally to the signal via their TCR.

Self-reactive T cells are activated by the 65-kDa mycobacterial heat-shock protein in neonatally thymectomized mice.

To elucidate the mechanism of autoimmune disease in neonatally thymectomized (NTX) mice, we have investigated the responsiveness of the self-reactive T cells which have not undergone clonal deletion in such animals. Consistent with a recent report (Yuuki et al., Eur. J. Immunol. 1990. 20: 1475), T cells bearing V beta 11-gene products capable of recognizing I-E-encoded molecules were readily detected in the mature T cell pool of NTX BALB/c (I-Ed, Mls-2a) mice. The V beta 11-bearing T cells in NTX mice expressed interleukin 2 receptors and responded normally to signals delivered through the T cell receptor. Notably, these T cells in NTX mice proliferated significantly after culture with the 65-kDa mycobacterial heat-shock protein, whose amino acid sequence is highly homologous to that in eukaryotes. These results suggest that self-reactive T cells in NTX mice may be activated by heat-shock proteins derived from various pathogens and/or stressed autologous cells, resulting in the development of autoimmune diseases in such animals.

Clonal anergy in self-reactive alpha/beta T cells is abrogated by heat-shock protein-reactive gamma/delta T cells in aged athymic nude mice.

Although T cells proliferate and differentiate primarily in the thymus, athymic nude mice contain an appreciable level of T cell receptor alpha/beta and gamma/delta T cells, suggesting the existence of the extrathymic pathway in the development of both T cells. Recent studies with nude mice indicate that clonal deletion of self-reactive T cells does not occur extrathymically. In the present study, we have investigated the responsiveness of self-reactive T cells differentiating along an extrathymic pathway in aged BALB/c (H-2d, Mls-1b2a, I-E+, 7-8 month old) nude mice. Consistent with recent reports, T cells bearing V beta 3 or V beta 11, which are important for recognizing proteins encoded by the Mls-2a or the I-E allele, respectively, are readily detected in age nude mice. The V beta 3- or V beta 11-bearing T cells, however, do not proliferate in response to staphylococcal enterotoxin A which specifically stimulates V beta 3- or V beta 11-bearing T cells. When exogenous recombinant interleukin 2 was added to the culture, the V beta 3-bearing T cells in aged nude mice significantly proliferated in response to staphylococcal enterotoxin A. Aged nude mice also contained a substantial level of gamma/delta T cells which account for 15.6% of all Thy-1.2+ cells. The gamma/delta T cells proliferated and produced a significant level of interleukin 2 in response to the 65-kDa mycobacterial heat-shock protein, which is highly homologous to its eukaryotic counterpart. These results suggest that unresponsiveness of self-reactive T cells may be reversed by T cells responding to stress proteins expressed by the invading microbes and/or the stressed autologous cells.

Sequential appearance of host-derived T cell subsets during differentiation in nude mice grafted with rat fetal thymus.

To elucidate the abnormality of T cell differentiation in nude mice grafted with rat fetal thymus that develop multiple-organ-localized autoimmune diseases, we examined sequential appearance of T cell subsets and expression of TCR genes in BALB/c nude mice after grafting with fetal F344 rat thymus. We observed progressive expression of TCR gamma/delta-alpha/beta genes in the lymph node (LN) cells from 8 to 12 wk after grafting. An appreciable number of CD4+ T cells but few CD8+ T cells were detected in the LN at 8 wk after grafting. CD8+ T cells increased slowly in number by 12 wk after grafting but remained at a low level in comparison with those in nude mice 12 wk after grafting with BALB/c thymus. In correlation with an increase in the number of T cells expressing TCR alpha/beta genes, alloreactivity as assessed by MLR was increased to a normal level. However, CTL activity against alloantigens remained at a low level in the LN cells at 12 wk. At this stage, organ-specific autoimmune diseases and a high level of anti-DNA autoantibodies were detected. In these mice host-reactive T cells such as V beta 3- or V beta 11-bearing T cells were virtually eliminated in the peripheral mature T cell pool, whereas T cells maturing in the fetal rat thymus significantly proliferated in response to donor-rat stimulator cells. These results suggest that the development of the autoimmune diseases may be ascribed to an impaired maturation of CD8+ T cells but not to failure in clonal elimination of host-reactive T cells in nude mice grafted with rat thymus.

Deletion of self-reactive T cells in nude mice grafted with neonatal allogeneic thymus.

The cellular basis of tolerance induction has been investigated in BALB/c(H-2d, thy 1.2, M1s1b2a) nude mice grafted with thymus of neonatal AKR/J mice(H-2k,Thy1.1,M1s1a2b). The spleen cells from nude mice grafted with AKR/J thymus showed a significantly decreased level of primary cytotoxic T cell response when stimulated with AKR/J cells, although these cells lysed well target cells of a third party C57BL/6 when stimulated with C57BL/6 cells. Consistent with CTL responses, T cells bearing V beta 6, that is important for recognizing M1s1a-encoded products of the thymic phenotype, were virtually abolished in the spleen and lymph node cells of nude mice 8 wk after grafting with AKR/J thymus. However, a substantial number of V beta 6-bearing T cells were detected in the peripheral organs of nude mice 23 wk after grafting with AKR/J thymus and in those of nude mice grafted with AKR/J fetal thymus depleted of macrophages/dendritic cells by incubating with 2'-deoxyguanosine in vitro before grafting. On the other hand, T cells bearing V beta 3, which are selectively related to M1s2a-encoded products of the host phenotype, were expressed neither on the peripheral T cells of nude mice grafted with AKR/J thymus at any stage after grafting nor on those of nude mice grafted with 2'-deoxyguanosine-treated AKR/J thymus. These data suggested that both V beta 6 and V beta 3 T cells were eliminated in the thymus of nude mice grafted with AKR/J thymus, presumably on the basis of interaction with both of graft-derived persisting and host-derived hemopoietic cells in the thymus and that thymic epithelium appears to have little capacity to eliminate T cells reactive to minor lymphocyte stimulating-encoded products.

The expression and sequences of T cell antigen receptor beta-chain genes in the thymus at an early stage after sublethal irradiation.

The sequential appearance of the thymocyte subpopulations and TCR gene messages occurred in the thymus of AKR mice (H-2k, Mlsa) from 7 to 14 days after sublethal irradiation. The thymocytes on day 7 after irradiation were composed of a large number of CD4+CD8+ blast-like cells and a relatively high proportion of CD4-CD8- cells (15 to 25%) but few CD3highCD4+CD8-/CD4-CD8+ cells. Approximately 22% of the CD4-CD8- cells were CD3high and -27% of the CD3highCD4-CD8- cells (-6% of whole CD4-CD8- cells) were F23.1+. The thymocytes on day 7 expressed a large amount of gamma- and delta-chain gene transcripts but reduced levels of alpha- and beta-chain gene transcripts. The V gene repertoire of 18 functional beta-chain cDNA derived from the thymocytes on day 7 was compared with those of 20 functional beta-chain cDNA derived from the thymocytes on day 14 which were composed of a large number of CD3lowCD4+CD8+ small-sized cells and a small number of CD3highCD4+CD8- cells. It is noteworthy that the distribution of V beta genes expressed in the thymocytes on day 7 was much the same as that in the thymocytes on day 14 but significantly different from that in normal BALB/c thymocytes as previously described. Interestingly, neither V beta 8.1 nor V beta 6 genes, which are important for recognition of the product of the Mlsa locus, was detected in these two cDNA libraries. These results suggest that clonal selection of TCR V beta repertoire, irrespective of positive or negative selection, appears to occur at the early stage of T cell differentiation, i.e., on the blast-like CD4+CD8+ thymocytes.

Functional T cell receptor delta chain gene messages in athymic nude mice.

The rearrangement and expression of T cell antigen receptor (TcR) delta chain genes were investigated in congenitally athymic nude mice. The lymphoid cells derived from nude mice showed evidence of rearranged delta chain genes and a relatively high level of delta chain gene messages. Nucleotide sequence analysis revealed that nude mice contained an in-frame delta chain transcript, composed of V delta 5-D delta 1-D delta 2-J delta 1-C delta genes in the spleen. We have previously described functionally rearranged TcR gamma chain genes in the spleen of nude mice. Taken together, T cell precursors appear to proliferate and differentiate along the extrathymic pathway into TcR gamma/delta-bearing T cells in nude mice.