Human thymic epithelium and T cell development: current issues and future directions

The human thymus develops early in fetal gestation with morphologic maturity reached by the beginning of the second trimester. TE3+ cortical thymic epithelium is most likely derived from endodermal third pharyngeal pouch, while A2B5/TE4+ medullary and subcapsular cortical thymic epithelium is likely derived from third pharyngeal cleft ectoderm. Fetal liver and yolk sac CD7+, CD4-, CD8-, surface(s) CD3- T cell precursors begin to colonize the thymus between 7 and 8 weeks of fetal gestation, followed by rapid expression of other T lineage surface molecules on developing thymocytes. CD7+, CD4-, CD8-, sCD3- thymocytes give rise to T cells of both the TCR alpha beta and TCR gamma delta lineages. Human thymic epithelial cells produce numerous cytokines including IL1, IL6, TGF alpha, leukemia inhibitory factor (LIF), M-CSF, G-CSF and GM-CSF- molecules that likely play important roles in multiple stages of thymocyte selection, activation and differentiation. Important areas for future research on human thymic epithelium include study of lymphoid and non-lineage differentiation potentials of CD7+, CD4-, CD8-, sCD3- T cell precursors in response to TE-cell produced cytokines, study of the triggering signals of cytokine release within the thymic microenvironment, and study of TCR-MHC mediated TE-thymocyte interactions.     

Forced expression of terminal deoxynucleotidyl transferase in fetal thymus resulted in a decrease in gammadelta T cells and random dissemination of Vgamma3Vdelta1 T cells in skin of newborn but not adult mice

The repertoire of lymphocyte receptor genes encoded in a germline is further diversified by a number of processes, including the template-independent addition of nucleotides (N regions) by means of terminal deoxynucleotidyl transferase (TdT). Normally, mouse gammadelta T cells in the early fetal thymus, whose T-cell receptor (TCR) genes lack N regions and are encoded by Vgamma3-Jgamma1 and Vdelta1-Ddelta2-Jdelta2 with canonical junctions (invariant Vgamma3Vdelta1), are thought to be the precursors of dendritic epidermal T cells (DETC). We generated mutant mice whose endogenous TdT promoter was replaced with the lck promoter through homologous recombination. These mutant mice expressed TdT in fetal thymus, had abundant N regions and infrequent canonical junctions in gamma and delta rearrangements, and showed a decreased number of gammadelta T cells. Various Vgamma3Vdelta1 T cells, most of which had N regions in their TCR genes, were found to disseminate in the skin of newborn mutant mice, whereas normal numbers of DETCs with the invariant Vgamma3Vdelta1 rearrangement were observed in adult mutants. These data demonstrate that the regulation of TdT expression during fetal development is important for the generation of gammadelta T cells, and that Vgamma3Vdelta1 T cells, which have various junctional sequences in their TCR genes, randomly disseminate in skin, but invariant Vgamma3Vdelta1 T cells have a great advantage for proliferation in skin.     

Abrogation of the Allelic Exclusion in a T Cell Receptor β Chain Gene Transgenic Mouse Strain

The expression of endogenous T cell receptor (TcR) β chains in a TcR β chain gene transgenic mouse (TGM) strain was examined. Unlike many other TGM strains reported, a considerable proportion of T cells from the thymus and spleen as well as organ cultured fetal thymus from our TGM express endogenous TCR β chains on their surface. Compatible with this was the elucidation of VDJ rearrangement of endogenous β chain genes by PCR. Three color flowcytometric analysis of thymus cell subpopulations revealed that the expression levels of both endogenous and transgenic TcR β genes are regulated in a maturational stage specific manner. Splenic T cells contained a several fold higher percentage of endogenous TcR β positive cells than thymus cells, suggesting a role of TcR on T cell peripherization. Vβ6 positive cells were deleted in the TGM carrying minor lymphocyte stimulating (Mls)-l^a antigen, indicating that the endogenous TcR β is functional in terms of transmiting a signal for clonal deletion.     

The myelin basic protein-specific T cell repertoire in (transgenic) Lewis rat/SCID mouse chimeras: preferential Vβ8.2 T cell receptor usage depends on an intact Lewis thymic microenvironment

In the Lewis rat, myelin basic protein (MBP)-specific, encephalitogenic T cells preferentially recognize sequence 68–88, and use the Vβ8.2 gene to encode their T cell receptors. To analyze the structural prerequisites for the development of the MBP-specific T cell repertoire, we reconstituted severe-combined immunodeficient (SCID) mice with fetal (embryonic day 15–16) Lewis rat lymphoid tissue, and then isolated MBP-specific T cell lines from the adult chimeras after immunization. Two types of chimera were constructed: SCID mice reconstituted with rat fetal liver cells only, allowing T cell maturation within a chimeric SCID thymus consisting of mouse thymic epithelium and rat interdigitating dendritic cells, and SCID mice reconstituted with rat fetal liver cells and rat fetal thymus grafts, allowing T cell maturation within the chimeric SCID and the intact Lewis rat thymic microenvironment. Without exception, the T cell lines isolated from MBP-immunized SCID chimeras were restricted by MHC class II of the Lewis rat (RT1.B¹), and none by I-A^d of the SCID mouse. Most of the T cell lines recognized the immunodominant MBP epitope 68–88. In striking contrast to intact Lewis rats, in SCID mice reconstituted by rat fetal liver only, MBP-specific T cell clones used a seemingly random repertoire of Vβ genes without a bias for Vβ8.2. In chimeras containing fetal Lewis liver plus fetal thymus grafted under the kidney capsule, however, dominant utilization of Vβ8.2 was restored. The migration of liver-derived stem cells through rat thymus grafts was documented by combining fetal tissues from wild-type and transgenic Lewis rats. The results confirm that the recognition of the immunodominant epitope 68–88 by MBP-specific encephalitogenic T cells is a genetically determined feature of the Lewis rat T cell repertoire. They further suggest that the formation of the repertoire requires T cell differentiation in a syngeneic thymic microenvironment.     

Vaccination with T cell receptor peptides primes anti-receptor cytotoxic T lymphocytes (CTL) and anergizes T cells specifically recognized by these CTL

We selected three peptides from the germ-line sequence of the Vβ8.2 and Jβ2.3 gene segments of the murine T cell receptor for antigen (TCR) which contained putative K^d- and L^d-restricted epitopes. Immunization of BALB/c (H-2^d) mice with the Vβ8.2(67–90) 23-mer peptide 1 as well as the 15-mer Vβ8.2(95–108)-peptide 2 efficiently primed specific CD8⁺ cytotoxic T lymphocyte (CTL) responses in vivo against natural TCR-Vβ8.2 epitopes. Vβ8.2⁺ T cells were not deleted in TCR peptide-immunized mice because the fractions of Vβ8.2⁺ CD4⁺ and Vβ8.2⁺ CD8⁺ T cells in spleen and lymph nodes were not altered. The proliferative response of Vβ8.2⁺ T cells to stimulation by monoclonal antibody F23.2 was selectively suppressed (by 60–80%) in peptide-immunized BALB/c mice, indicating partial anergy of this T subset. Immunization of BALB/c mice with the Jβ2.3-derived peptide 3 stimulated a CD8⁺ CTL response against a class I-restricted epitope within this Jβ segment that was also generated during natural “endogenous” processing of this self antigen. These data confirm the predictive value of major histocompatibility complex class I allele-specific motifs. The described experiments indicate that TCR peptide-primed CD8⁺ CTL recognize class I-restricted, natural Vβ/Jβ-TCR epitopes. Such anti-TCR CTL may, thus, operate in Vβ-specific immunoregulation of the T cell system suppressing their functional reactivity without deleting them.     

Developmentally regulated fetal thymic and extrathymic T-cell receptor gamma delta gene expression

The gamma delta T-cell receptor (TCR) is the first TCR to be expressed in ontogeny in all vertebrates in which it has been examined thoroughly. Murine gamma delta cell-surface protein is detected by the fourteenth day of gestation. In this work, the activation of gamma delta RNA has been studied. Data indicate that the first TCR protein to appear in the thymus is encoded by gamma genes that are activated after cells colonize the thymus. However, the sequential appearance of different gamma delta TCR proteins during thymic ontogeny cannot be readily explained by differential temporal activation of V gamma genes in the thymus. There are distinct patterns of gamma and delta gene expression during fetal liver development and in the fetal gut (or tissue associated with it). Cells apparent in the liver of mice at birth express gamma delta cell-surface protein, but they disappear from the liver very soon afterward. One V gamma gene is rearranged and expressed prethymically. In addition, gamma gene expression is detectable in the livers of newborn athymic mice. Together, these observations indicate a thymic-independent pathway of activation of TCR genes.     

Partial reconstitution of V(D)J rearrangement and lymphocyte development in RAG-deficient mice expressing inducible, tetracycline-regulated RAG transgenes

Previously, we described a tetracycline-based autoregulatory system for inducible gene expression in mammalian cells and transgenic mice [Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 6522]. We have tested the ability of this system to drive functional expression in vivo of the V(D)J recombination activating genes, RAG1 and RAG2. In induced transgenic mice, transgenic RAG1 and RAG2 mRNA is observed in thymus and spleen, and expression of both transgenes on the RAG1 or RAG2 knockout backgrounds allows partial, inducible, lymphocyte reconstitution. In thymus and peripheral lymphoid organs of reconstituted animals, cells expressing CD4 and/or CD8 on their surface, also express CD3 and TCR beta chain. In these animals, V(D)J rearrangements are detected in thymus, lymph nodes, and spleen at the TRB locus, and in thymus and lymph nodes at the TRD locus. At the TRA locus, broken ends at V(D)J recombination signals are detected only in thymus, as are reciprocal signal joint products derived from deletional rearrangement. T cell reconstitution occurs in these animals whether they are induced in utero during development, or shortly after birth. A low level of B cell reconstitution is also observed. B220+IgM+ cells are observed in spleen only in induced animals, and rearrangements at IGH and IGK loci are detected in bone marrow and spleen. Broken signal ends at the IGK locus, are not detected in peripheral lymphoid organs. Inducible reconstitution of normal levels of serum immunoglobulin, including heavy chain class switch isotype variants is also observed in these animals. Further, these transgenes do not appear to interfere with lymphocyte development mediated by functionally rearranged TRB chain or IGH chain transgenes in RAG-deficient animals. These mice provide a unique system for the inducible activation of V(D)J recombination and the development of primary lymphocytes.     

Analysis of TCR β chains in Lewis rats with experimental allergic encephalomyelitis. II. Vβ8.2+ T cells with limited CDR3 N region additions derive from the adult thymus

Immunization of Lewis (LEW) rats with guinea pig myelin basic protein (MBP) induces a population of encephalitogenic CD4 T cells having specificity for the dominant immunogenic peptide of MBP, 68 – 86. The TCR β chains of these disease-causing T cells show three distinct features: they are almost exclusively Vβ8.2, they use AspSer as the first two amino acid residues of the third complementarity-determining region (CDR3) and these junctional region sequences show few if any non-germline N-region nucleotide additions. This last feature raises the possibility that these autoimmune T cell precursors derive from TCR gene rearrangements occurring during early, perinatal ontogeny, a period when the enzyme terminal deoxynucleotidyl transferase (TdT), responsible for N region additions, is not expressed. An alternative possibility is that these features of the TCR of MBP 68 – 86-reactive T cells are dictated by considerations of antigen selection throughout ontogeny both in the thymus and in the periphery – i.e., that such β chains are conformationally the most appropriate for triggering by an epitope of 68 – 86 complexed to class II RT1.B^l MHC molecules. We show here that active experimental allergic encephalomyelitis, while delayed in onset, occurs in heavily irradiated animals, but not in the absence of a thymus, a finding indicating that this autoimmune disease is caused by a T cell subpopulation derived from the post-irradiation adult thymus. These disease-causing T cells are heavily Vβ8.2⁺ , CDR3 AspSer⁺ and use few N region additions. We conclude that T cells with these TCR β chain features can be generated in the adult thymus and most likely reflect requirements imposed by antigen selection.