Cross-lineage expression of Ig-beta (B29) in thymocytes: positive and negative gene regulation to establish T cell identity

Abstract

Developmental commitment involves activation of lineage-specific genes, stabilization of a lineage-specific gene expression program, and permanent inhibition of inappropriate characteristics. To determine how these processes are coordinated in early T cell development, the expression of T and B lineage-specific genes was assessed in staged subsets of immature thymocytes. T lineage characteristics are acquired sequentially, with germ-line T cell antigen receptor-beta transcripts detected very early, followed by CD3epsilon and terminal deoxynucleotidyl transferase, then pTalpha, and finally RAG1. Only RAG1 expression coincides with commitment. Thus, much T lineage gene expression precedes commitment and does not depend on it. Early in the course of commitment to the T lineage, thymocytes lose the ability to develop into B cells. To understand how this occurs, we also examined expression of well defined B lineage-specific genes. Although lambda5 and Ig-alpha are not expressed, the mu 0 and I mu transcripts from the unrearranged IgH locus are expressed early, in distinct patterns, then repressed just before RAG1 expression. By contrast, RNA encoding the B cell receptor component Ig-beta was found to be transcribed in all immature thymocyte subpopulations and throughout most thymocyte differentiation. Ig-beta expression is down-regulated only during positive selection of CD4(+)CD8(-) cells. Thus several key participants in the B cell developmental program are expressed in non-B lineage-committed cells, and one is maintained even through commitment to an alternative lineage, and repressed only after extensive T lineage differentiation. The results show that transcriptional activation of "lymphocyte-specific" genes can occur in uncommitted precursors, and that T lineage commitment is a composite of distinct positive and negative regulatory events.

Figure 1

Summary of immature cell populations in the immunodeficient mouse thymus and their developmental relationships. Bold type indicates the names of subsets of cells analyzed in this paper. The extended profiles of phenotypic markers are from refs. and and H.W., R.A.D., and E.V.R., unpublished results. Italic designations A–D key these populations to the samples studied in Fig. 2. Cell fates and developmental relationships among populations A through D are based on work by others using the corresponding populations from normal adult thymus, in which populations A and B are not separated (11, 12). The CD44⁺ CD25⁺ subset of HSA⁺ cells (population C) has lost the potential to give rise to B or NK cells, although it retains the ability to give rise to dendritic cells (not shown). Cells beyond this stage (e.g., population D) are fully T lineage committed. The possibility that the NK-like cells (population B) can give rise to T cells as well as NK cells is based on the properties of a similar subset in fetal thymus (–53). The CD4⁺ subset, which has been studied extensively in normal thymus, includes a subset of population A plus some of the transitional cells between A and C that have not yet acquired CD25 (H.W., R.A.D., and E.V.R., unpublished results).

Figure 2

Expression of early T cell genes in immature thymocytes and normal fetal liver cells. RT-PCR was performed with the indicated primers to compare subsets of cells from immunodeficient mouse thymocytes with subsets from wild-type C57BL/6 fetal liver. All samples presented in one lane were derived from equal aliquots of the same cDNA sample. Subpopulations analyzed in each lane are indicated by the letters under each sample. For thymocyte subsets: U, unfractionated; A, HSA^–Sca-1⁺; B, HSA^– Sca-1^–; C/D, HSA⁺ (Sca-1⁺). For fetal liver subsets: T, total; N, B220⁻ Sca-1⁻ Gr-1⁻ (triple negative); L, B220⁺ Sca-1⁺ Gr-1⁻ (lymphoid-enriched); S, B220⁻ Sca-1⁺ Gr-1⁻ (stem cell enriched); G, Sca-1⁻ Gr-1⁺ cells. (A) Lanes: 1–4, RAG2^−/− thymus; 5–8, C.B 17-scid/scid thymus; 9–13, C57BL/6 fetal liver subsets. (B) Lanes: 1–4, B6-scid/scid thymus; 5–8, C57BL/6 fetal liver. RAG1 levels are higher in RAG-2^−/− thymocytes than in SCID thymocytes because the former survive longer after the induction of recombinase expression (13). TdT is expressed only during postnatal lymphocyte development.

Figure 3

Preferential expression of Ig-β (B29) in populations containing B cells and thymocytes. (A) Analysis of Ig-β RNA expression in different tissues. Lanes: 1, skeletal muscle; 2, T enriched spleen (>93% T cells); 3 and 6, normal mouse splenocytes (≥60% B cells; 4, total C57BL/6-scid/scid thymocytes; 5, normal C57BL/6 thymocytes. (B) Comparison of Ig-β and Iμ expression in subsets of normal mouse splenic T cells and B cells. T cell subsets were purified as described in Materials and Methods. Samples without reverse transcriptase gave no signal (data not shown). (Top) Products of 33 PCR cycles detected by ethidium bromide staining. (Bottom) Products of 24 PCR cycles detected by hybridization. Lanes: 1, CD4⁺ T cells; 2, CD8⁺ T cells; 3, T enriched spleen; 4, total spleen; 5, B220⁺ B enriched cells.

Figure 4

Shutoff of Ig-β expression at a late stage in the positive selection of CD4⁺ CD8⁻ thymocytes. (A) Summary of phenotypic changes accompanying positive selection. (B and C) Three-color flow cytometry of normal C57BL/6 thymocytes stained for CD4, CD8, and HSA expression (B) or for CD4, CD8, and CD69 expression (C). The sorting gates used in the two sorting experiments to dissect positive selection stages are indicated, as described in Materials and Methods. (D and E) RT-PCR analyses of RNA levels in the populations fractionated from the stained samples in B (D) and in C (E). Also shown (bottom row, D and E) are autoradiograms of the hybridization of an internal probe to the Ig-β PCR products from these samples. Note that in E, the samples in lanes 5 (CD4⁺ CD8⁻ CD69⁻) and 6 (CD4⁺ CD8⁻ CD69⁺) are reversed with respect to their order in development.