Early growth response (Egr)-1 gene induction in the thymus in response to TCR ligation during early steps in positive selection is not required for CD8 lineage commitment.

The early growth response gene 1 (Egr-1) is induced during positive selection in the thymus and has been implicated in the differentiation of CD4+ thymocytes. Here, we show that signals that specifically direct CD8 lineage commitment also induce Egr-1 DNA-binding activity in the nucleus. However, we find that pharmacological inhibition of mitogen-activated protein kinase/extracellular signal-related kinase kinase activity potently inhibits Egr-1 DNA-binding function at concentrations that promote differentiation of CD8+ thymocytes, suggesting Egr-1 activity is not essential for CD8 commitment. To further determine the role of Egr-1 in thymocyte development, we compare steady-state Egr-1 DNA-binding activity in thymocytes from mice with defined defects in positive selection. The data indicate that the appearance of functional Egr-1 is downstream of signals induced by TCR/MHC engagement, whereas it is less sensitive to alterations in Lck-mediated signals, and does not correlate directly with proficient positive selection. Egr-1 is one of the earliest transcription factors induced upon TCR ligation on immature thymocytes, and plays a potential role in the transcription of genes involved in thymocyte selection.

Tolerance induction by elimination of subsets of self-reactive thymocytes.

Immature thymocytes expressing TCRs which confer reactivity to self-MHC molecules are subject to efficient elimination as a result of negative selection. Previously, we have identified a lineage of H-2Kb Tg mice, CD2Kb-3, which fails to reject skin grafts from mice expressing H-2Kb even though H-2Kb-specific cytotoxic T cells can be generated in vitro. We now show that bone marrow derived cells are responsible for tolerance induction and that tolerance is acquired, at least in part, by negative selection in CD2Kb-3 mice. Thymocytes expressing two different transgenic TCR (TCR-Tg) clonotypes conferring reactivity to H-2Kb are eliminated prior to the CD8+CD4+ stage of differentiation in double Tg (CD2Kb-3 x TCR-Tg)F1 mice. As in other cases where thymocytes from TCR-Tg mice develop in the presence of deleting ligands, large numbers of TCR+ CD8-CD4- T cells accumulate in double Tg mice. However, these T cells fail to respond to H-2Kb in vitro but can be activated with immobilized anti-clonotypic antibody. Consequently, thymocytes expressing these types of TCR molecules represent a fraction of H-2Kb-reactive thymocytes which are unable to mature into T cells capable of mounting H-2Kb-specific cytotoxic responses. Presumably, precursors of H-2Kb-specific cytotoxic T cells found in the periphery of CD2Kb-3 mice express a distinct repertoire of TCR molecules conferring reactivity to H-2Kb. We consider potential explanations to account for this discrepancy and their wider implications, including the possibility that the repertoire of thymocytes able to recognize self-H-2Kb molecules in CD2Kb-3 mice is divided into distinct subsets; those which are, and those which are not, subject to negative selection.

Transcription of HLA-G transgenes commences shortly after implantation during embryonic development in mice.

We studied the pattern of transcription of a human HLA-G transgene in mice using polymerase chain reaction (PCR) techniques. Transcription of the HLA-G transgene commenced in cells derived from embryos as soon as 48 hr after implantation of embryos in the uterine wall and continued for at least a further 48 hr during embryonic development. HLA-G transcripts were also present in RNA extracted from thymus, spleen and liver of adult HLA-G transgenic mice, although transcripts were not detected in RNA from any other tissues except testes of male transgenic mice. These results demonstrate that the restricted pattern of HLA-G transcription in embryo-derived trophoblast cells during the first trimester of human pregnancy is reproducible in mice. This suggests that transcription factors required for a highly regulated pattern of gene expression during embryonic development are present in murine trophoblast cells and provide a means to investigate the factors and study the consequences of HLA-G expression during development of the embryo.

Induction of tolerance to self MHC class I molecules expressed under the control of milk protein or beta-globin gene promoters.

We have studied tolerance induction in transgenic CBA mice expressing H-2Kb genes under the influence of guinea-pig alpha-lactalbumin (KAL) or human beta-globin gene promoter (K beta). KAL radio-resistant cells, but not bone marrow derived cells, induce tolerance to H-2Kb in chimeric mice. In contrast, bone marrow derived and radio-resistant cells of K beta mice induce tolerance. Although appropriate, tissue-specific, expression of H-2Kb molecules occurs in KAL and K beta mice, H-2Kb is expressed at low levels in thymus of transgenic mice. In addition, dendritic cells and macrophages express H-2Kb molecules when K beta, but not when KAL bone marrow is cultured in vitro. The mode of tolerance induction was examined in double transgenic mice by mating KAL or K beta mice to mice expressing TCR transgenes (Tg-TCR) derived from a H-2Kb specific, CD8-independent cytotoxic T cell clone. In both cases, a large number of Tg-TCR+ CD8+CD4+ thymocytes develop but mature CD8+CD4- thymocytes fail to appear suggesting that thymocytes are eliminated late in development. Some CD8-CD4- and CD8-CD4+ Tg-TCR+ T cells develop in double transgenic mice and respond to activation through their TCR-CD3 complex in vitro, although no responses to stimulation with H-2Kb expressing cells were detected. Thus, tolerance induction in KAL and K beta mice proceeds via a deletional mechanism that is inefficient due either to low numbers of H-2Kb expressing thymic cells or to the low levels of H-2Kb expressed by thymic cells, or to a combination of these factors.

Expression and function of Qa-2 major histocompatibility complex class I molecules in transgenic mice.

Qa-2 molecules are weak transplantation antigens encoded by class I genes of the major histocompatibility complex. When expressed in transgenic CBA mice, Qa-2 molecules provoke rapid rejection of skin grafts and strong, Qa-2 specific, cytotoxic T-cell responses. Efficient rejection of skin grafts from Qa-2 transgenic mice takes place when Qa-2 molecules are attached to the cell membrane with a glycophosphatidyl anchor or by a transmembrane protein domain, except that rejection times are slightly longer in the former case. These results demonstrate that Qa-2 molecules can behave as major transplantation antigens, as do closely related H-2 molecules. Failure of Qa-2 molecules to provoke strong T-cell responses in non-transgenic mice is probably due to the very low level of expression of Qa-2 molecules in skin keratinocytes from such mice since these cells express increased levels of Qa-2 molecules in all Qa-2 transgenic mice.