Thymocyte maturation is regulated by the activity of the helix-loop-helix protein, E47.

The E2A proteins, E12 and E47, are required for progression through multiple developmental pathways, including early B and T lymphopoiesis. Here, we provide in vitro and in vivo evidence demonstrating that E47 activity regulates double-positive thymocyte maturation. In the absence of E47 activity, positive selection of both major histocompatibility complex (MHC) class I- and class II-restricted T cell receptors (TCRs) is perturbed. Additionally, development of CD8 lineage T cells in an MHC class I-restricted TCR transgenic background is sensitive to the dosage of E47. Mice deficient for E47 display an increase in production of mature CD4 and CD8 lineage T cells. Furthermore, ectopic expression of an E2A inhibitor helix-loop-helix protein, Id3, promotes the in vitro differentiation of an immature T cell line. These results demonstrate that E2A functions as a regulator of thymocyte positive selection.

Interleukin 7 induces TCR gene rearrangement in adult marrow-resident murine precursor T cells.

Rearrangement of the T cell antigen receptor genes is a complex, highly regulated process. To gain a better understanding of the extracellular factors involved in the regulation of TCR beta and gamma gene rearrangement in adult murine bone marrow-resident precursor T cells, several cytokines were tested for their ability to induce gene recombination. A selected population of C58/J bone marrow cells (Thy 1(low), CD3, CD8, B220) that is enriched for pre-T cell activity was propagated in vitro in medium supplemented with IL-3 and mast cell growth factor (MGF, also referred to as stem cell factor, Steele factor and c-kit ligand). These cytokines were required for the maintenance of pre-T cell activity in culture, but had no effect on TCR gene expression. Several additional cytokines were added to the culture medium. Of all those tested, only IL-7 induced complete rearrangement of the TCR gamma locus. Complete rearrangement of the TCR beta locus was not induced under any of the culture conditions analysed here. The bone marrow cells cultured in IL-3, MGF and IL-7 did not begin to express mature T cell proteins and maintained their in vivo progenitor potential. Furthermore, IL-7 cultured bone marrow cells were capable of differentiation in vivo into all phenotypic subpopulations of T cells, without an apparent bias toward the gammadelta lineage. The data presented here suggest that TCR gamma gene rearrangement in adult pre-T cells is regulated by IL-7, but that the TCR beta locus requires additional or alternative signals for the induction of complete rearrangement.

Transcription of the TCR-beta locus initiates in adult murine bone marrow.

Successful expression of the TCR beta-chain gene is a multistep process that involves: 1) initial transcription of multiple, unrearranged gene segments, 2) rearrangement of V, D, and J gene segments to form a complete beta-chain gene, and 3) transcription of the fully rearranged beta gene. All of these events have been shown to occur in the thymus, where the majority of T cell development takes place; however, the extent to which any of these events may occur prethymically has not been established. To examine prethymic TCR-beta gene expression, RNA was isolated from a precursor T cell-enriched population (Thy 1low CD3-) of C58/J mouse bone marrow, and analyzed by reverse transcriptase-PCR. A transcript containing TCR-beta constant (C) region sequences but not variable (V) region sequences was amplified, suggesting that an unrearranged TCR-beta gene locus is transcriptionally active in this bone marrow population. The same product was detected in Thy 1+ CD3- bone marrow cells from nude mice, indicating that the thymic microenvironment is not necessary for initiation of TCR-beta gene transcription. This C beta transcript is not confined to pre-B cells, as it was identified in RNA isolated from Thy 1low CD3- B220- bone marrow cells. Germline V beta transcripts were also detected in RNA from this bone marrow population. Furthermore, Sca-1+ Lin- and Sca-1+ Lin+ bone marrow populations from both C58/J mice and nude mice also expressed the C beta transcript. DNA-PCR analyses with D beta-J beta primer sets revealed that partial rearrangement of the beta locus had occurred in all bone marrow populations analyzed. These data suggest that both transcription and partial rearrangement of the TCR-beta locus can initiate in bone marrow cells of adult mice, before exposure of these cells to the thymus.

In vitro growth of bone marrow-resident T cell precursors supported by mast cell growth factor and IL-3.

The growth requirements of bone marrow-resident cells that are able to differentiate along the T cell lineage (pre-T cells) have not been well established. We recently have shown that the T cell-derived lymphokine IL-3 is able to maintain pre-T cells in vitro for at least 2 weeks. However, in our initial studies, we were not able to ascertain whether IL-3 induced pre-T cell growth during culture, or whether IL-3 simply maintained the viability of these progenitors. To address this issue, we used a multiple dose assay system to assess the level of pre-T cell activity (thymic repopulation) in a selected population of bone marrow cells (CD3-, Thy-1.2+) both before and after culture in IL-3. In addition, we tested the potential role of mast cell growth factor (MGF) in the growth and maintenance of pre-T cells in vitro. The results of these studies showed that IL-3 produced a modest, but consistent increase in the pre-T cell activity during culture. Culture of CD3-, Thy-1.2+ bone marrow cells in MGF also resulted in an increase in the total amount of detectable pre-T cell activity among the cultured cells. The most dramatic increases in pre-T cell activity, however, were induced by the culture of the selected marrow cells in both MGF and IL-3. Cultures supplemented with both cytokines produced net increases in pre-T cell activity of 40- to 75-fold after 10 days of culture. Because the increases in pre-T cell activity were not accompanied by observable increases in the size of thymic colonies produced by the pre-T cells, the increased levels of pre-T cell activity appeared to result from increases in pre-T cell numbers during culture. Thus, in addition to the other activities ascribed to MGF, this cytokine displays pre-T cell growth factor activity and can synergize with IL-3 in that capacity. The use of MGF in conjunction with IL-3 provides the best system described to date for the propagation of pre-T cells in primary bone marrow cell cultures.

Characterization of the progeny of precursor-T (pre-T) cells maintained in vitro by interleukin-3 (IL-3). Development of T-cell function in vivo.

We have recently described a bone marrow culture system which is able to maintain a portion of the precursor-T (pre-T) cell compartment of adult murine marrow in vitro, in the presence of interleukin-3 (IL-3), for at least 2 weeks. However, because growth in IL-3 might also induce the differentiation of the pre-T cells, it is necessary to determine the extent to which the developmental potential of the pre-T cells is altered during their residency in vitro. Previously, we analysed the progeny of cultured pre-T cells and compared their intrathymic development, their appearance in the periphery, and their V beta gene utilization to that of the progeny of fresh pre-T cells. Within these parameters, the cells derived from cultured marrow cells did not differ significantly from cells derived from fresh marrow cells. However, these studies did not allow us to determine the functional status of the T-cell progeny of cultured marrow. In the work presented here, we analysed the functional potential of T cells which were derived either from fresh pre-T cells or pre-T cells which had been maintained for 1 week in vitro. The T-cell mediated functions analysed included mitogen- and alloantigen-induced proliferation, IL-2 production, and generation of cytotoxic T cells. We found that the cultured pre-T cells were capable of giving rise to mature, immunocompetent T cells which did not differ significantly from the progeny of fresh pre-T cells in their functional potential.

Characterization of the progeny of pre-T cells maintained in vitro by IL-3: appearance in the periphery and V beta utilization in vivo.

We have recently demonstrated that bone marrow-resident cells, which are able to repopulate the thymus of irradiated recipient mice (pre-T cells), can be maintained in vitro for at least 2 weeks in the presence of exogenous IL-3. Because this marrow culture system can be applied to the study of early T cell differentiation, it is important to ascertain the extent to which in vitro culture of the pre-T cells might alter the T cell progeny which can develop from them. In previous work, we showed that the progeny of cultured pre-T cells appeared to develop in a kinetically normal fashion within the thymus of recipients and that the acquisition of key developmental markers (IL-2R and CD3) was identical in the progeny of fresh and cultured pre-T cells. Here, we report the results of experiments carried out to characterize the progeny of cultured pre-T cells which were found in the peripheral lymphoid tissues several weeks following intrathymic transfer to irradiated recipients. We found no remarkable differences between the progeny of cultured or fresh marrow cells with respect to the timing of their appearance in the periphery nor their expression of CD4 or CD8. By studying the patterns of utilization of five different V beta gene products by the T cells derived from fresh or cultured bone marrow, we were able to test the susceptibility of both sets of progeny to both positive and negative selection pressures during their in vivo maturation. These experiments established that the progeny of cultured marrow cells were equally susceptible to TCR repertoire selection, as were the progeny of fresh bone marrow cells, and that the process of in vitro growth did not alter the potential TCR repertoire of the pre-T cells.

Characterization of the progeny of pre-T cells maintained in vitro by IL-3: expression of the IL-2 receptor and CD3 during thymic development.

We have recently described a bone marrow culture system which is able to maintain, for at least 2 weeks, cells which have the capacity to repopulate the thymus of irradiated recipient mice (pre-T cells). Because this culture system depends upon the addition of an exogenous growth factor (IL-3) which may potentially influence the differentiation of the cultured pre-T cells, it is important to determine whether or not the progeny of cultured marrow cells are able to develop within the thymus in a kinetically normal fashion. Here we report the results of an analysis of the progeny of those cultured progenitor cells at 2, 3, and 4 weeks following intrathymic transfer. The passage of cultured donor-derived cells through critical early (expression of the IL-2 receptor) and late (expression of high levels of CD3) intrathymic events was assessed in these studies and compared with the pattern observed in the progeny of fresh bone marrow cells. The results of these studies showed that the progeny of cultured pre-T cells were able to develop expression of the IL-2 receptor and CD3 surface antigen during their residency within the thymus. In addition, both the timing and levels of expression of these surface markers were virtually identical on the progeny of fresh and cultured pre-T cells. These data suggest that cultured pre-T cells are not dramatically altered by their passage in vitro and are able to give rise to normally developing thymocytes upon in vivo transfer.