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Background & Aims: Syngeneic bone marrow transplantation into the adult tg@e26 mouse results in severe wasting disease and colitis. On transplantation, the rudimentary thymus of the adult tg@e26 mouse repopulates briefly but quickly regresses in temporal association with the onset of colitis. The aim of this study is to test the hypothesis that treatment of colitis restores thymic capability to generate regulatory T cells (CD4^+25^+ T"R cells). Methods: Colitis was induced by bone marrow transplantation into adult irradiated tg@e26 mice. Colitis was prevented by 3 distinct modalities. CD4^+25^+ T cells were collected from both the thymus and spleen and studied for regulatory function by an in vitro suppression assay. Results: CD4^+25^+ T"R cells do not develop efficiently in the thymus of bone marrow transplanted adult tg@e26 mice, which have a developmental arrest affecting thymic epithelium. By contrast, the thymic epithelium of neonatal tg@e26 mice supports development of T"R cells. Consequently, colitis develops only in adult transplanted mice but not in the neonatally transplanted mice. Treatment of colitis prevents destruction of the thymus of adult transplanted tg@e26 mice and T"R cells are produced. Conclusions: The negative impact of colitis on T"R development in the thymus, which was confirmed in a second model of colitis, has profound implications for the pathogenesis and treatment of human inflammatory bowel disease.

Background &Aims:: Tumor necrosis factor (TNF) induces multiple effects including cell proliferation and death by ligation with TNF receptor type II (TNFR2). We studied the role of TNFR2 in chronic inflammation-induced colonic epithelial alteration. Methods: TNFR2 expression in colonic epithelial cells (CECs) was assessed by ribonuclease protection assay (RPA) and immunohistochemistry (IHC) in patients with inflammatory owel disease (IBD) and murine colitis models. TNFR2 expression was also analyzed using COL0205 cells. The role of TNFR2 in colonic epithelial homeostasis was examined by generating interleukin 6-deficient TCR@aKO (@aIL-6DKO) or TNFR2- deficient TCRot (otTNFR2DKO) mice. Methods:: TNFR2 expression was up-regulated in CEC in both human ulcerative colitis and Crohn's disease. In vitro studies showed that TNFR2 expression was upregulated by a cooperative effect of key proinflammatory cytokines. By RPA, the increased expression of TNFR2 was detectable in TCR@aK0 mice with colitis compared with TCRotKO mice without colitis or wild-type mice. In @aIL-6DK0 mice, TNFR2 expression, proliferation, and nuclear factor kappa B activation of CECs were markedly reduced compared with TCRotKO mice. @aTNFR2 mice also showed significantly less colonic epithelial proliferation compared with TCRotKO mice. Conclusions:: Expression of TNFR2 is consistently increased on CECs in both murine colitis models as well as patients with IBD. TNFR2 may play an important role in colonic inflammation-associated alteration in the intestinal epithelium.

SAP, the gene that is altered or absent in the X-linked lymphoproliferative syndrome (XLP), encodes a small protein that comprises a single SH2 domain and binds to the cell-surface protein SLAM which is present on activated or memory T and B cells. Because defective NK cell activity also has been reported in XLP patients, we studied the SAP gene in NK cells. SAP was induced upon viral infection of SCID mice and shown to be expressed in NK cells by in vitro culturing in the presence of IL-2. Moreover, SAP was expressed in the NK cell lines YT and RNK 16. Because SLAM, the cell-surface protein with which SAP interacts, and 2B4, a membrane protein having sequence homologies with SLAM, also were found to be expressed on the surfaces of activated NK and T cell populations, they may access SAP functions in these populations. Whereas we found that 2B4 also binds SAP, 2B4-SAP interactions occurred only upon tyrosine phosphorylation of 2B4. By contrast, SLAM-SAP interactions were independent of phosphorylation of Y281 and Y327 on SLAM. As CD48, the ligand for 2B4, is expressed on the surface of Epstein-Barr virus (EBV)-infected B cells, it is likely that SAP regulates signal transduction through this pair of cell-surface molecules. These data support the hypothesis that XLP is a result of both defective NK and T lymphocyte responses to EBV. The altered responses may be due to aberrant control of the signaling cascades which are initiated by the SLAM-SLAM and 2B4-CD48 interactions.

The TCR-associated protein CD3ζ plays a major role in regulating the state of responsiveness to peptide-MHC complexes on the surface of antigen-presenting cells. In this paper the requirement of CD3ζ in the generation of cytotoxic T cells was compared with its requirement in cytokine gene activation in two mutant mice: ZKO mice with a disrupted CD3ζ gene and ZTG mice in which a truncated CD3ζ segment was expressed as a transgene on the ZKO background. Upon infection of ZTG mice with lymphocytic choriomeningitis virus (LCMV), antigen-specific cytotoxic T lymphocyte (CTL) responses were detected, identical to responses in wild-type mice. In addition, antigen-specific CTL responses to allogeneic class I and class II MHC in ZTG animals were indistinguishable from those in wild-type animals. However, CTL responses to the same major antigens were not detectable in ZKO mice. We conclude that the signal transduction pathways leading to CTL development and cytokine production can be triggered through TCR in the absence of functional CD3ζ, provided the remainder of the TCR-CD3 complex is expressed at high levels on the cell surface. Surprisingly, IFN-γ production in response to LCMV followed the same kinetics in ZKO, ZTG and wild-type mice. However, in vitro studies showed that cytokine production in general was abnormally regulated in T lymphocytes from ZKO mice, in contrast to ZTG T cells. Taken together, these studies support the hypothesis that development of CTL can take place in the absence of functional CD3ζ. However, CTL development requires stronger TCR-initiated signal transduction events than induction of cytokine genes.

We have examined the ability of the CD3-gamma delta epsilon and CD3-zeta signaling modules of the T cell receptor (TCR) to couple CD38 to intracellular signaling pathways. The results demonstrated that in TCR+ T cells that express the whole set of CD3 subunits CD38 ligation led to complete tyrosine phosphorylation of both CD3-zeta and CD3-epsilon polypeptide chains. In contrast, in TCR+ cells with a defective CD3-zeta association CD38 engagement caused tyrosine phosphorylation of CD3-epsilon but not of CD3-zeta. Despite these differences, in both cell types CD38 ligation resulted in protein-tyrosine kinase and mitogen-activated protein kinase activation. However, in cells expressing chimerical CD25-zeta or CD25-epsilon receptors or in a TCR-beta- Jurkat T cell line, CD38 ligation did not result in tyrosine phosphorylation of the chimeric receptors, or CD3 subunits, or protein-tyrosine kinase or mitogen-activated protein kinase activation. In summary, these results support a model in which CD38 transduces activating signals inside the cell by means of CD3-epsilon and CD3-zeta tyrosine phosphorylation. Moreover, these data identify the CD3-gamma delta epsilon signaling module as a necessary and sufficient component of the TCR/CD3 complex involved in T cell activation through CD38.

The TCR-associated CD3 complex consists of four subunits, i.e. CD3γ, δ, ε and ζ, which are expressed very early in T cell development prior to the expression of the TCR and the pre-TCR α chain. It is unclear whether the expression of each CD3 protein is independent of, or is influenced by, other CD3 subunits. T study whether CD37egr; regulates expression of CD3γ and δ genes, we generated a strain of CD3ε-deficient mice termed CD3ε^ΔP/ΔP (ε^ΔP), in which the promoter of CD3ε was disrupted, and subsequently reconstituted these mice with a CD3ε transgene. In the ε^ΔP mice, T cell development is arrested at the double-negative stage and targeting the CD3ε gene caused severe inhibition of CD3γ and δ gene expression. Introduction of the CD3ε transgene did not restore CD3γ and δ expression. However, a very small fraction of prothymocytes that expressed CD3γ and δ was rescued upon reconstitution of the CD3ε transgene. Remarkably, this rescue led to a very efficient differentiation and maturation of thymocytes, resulting in a significant T cell population in the periphery. These results demonstrate that CD3ε does not regulate expression of CD3γ and δ genes, and underscore the capacity of each prothymocyte to give rise to a large number of mature peripheral T cells.

The gene encoding the CD3‐epsilon chain of the T cell receptor (TCR/CD3) complex is uniquely transcribed in all T lymphocyte lineage cells. The human CD3‐epsilon gene, when introduced into the mouse germ line, was expressed in correct tissue‐specific fashion. The gene was then screened for T lymphocyte‐specific cis‐acting elements in transient chloramphenicol transferase assays. The promoter (‐228 to +100) functioned irrespective of cell type. A 1225 bp enhancer with strict T cell‐specificity was found in a DNase I hypersensitive site downstream of the last exon, 12 kb from the promoter. This site was present in T cells only. The CD3‐epsilon enhancer did not display sequence similarity with the T cell‐specific enhancer of CD3‐delta, a related gene co‐regulated with CD3‐epsilon during intrathymic differentiation. The CD3‐epsilon enhancer was unusual in that it constituted a CpG island, and was hypomethylated independent of tissue type. Two HTLV I‐transformed T cell lines were identified in which the CD3‐epsilon gene was not expressed, and in which the enhancer was inactive.

Clonally distributed (clonotypic) antigen receptors on human T lymphocytes (alpha and beta chains) are associated with three invariable T3 polypeptide chains (T3 gamma, delta and epsilon), together forming the T3/T cell receptor complex. Monoclonal antibodies prepared against the two 20‐kd T3 polypeptide chains demonstrated that T3‐delta and T3‐epsilon are distinct polypeptide chains. Only one monoclonal antibody (anti‐T3‐delta chain) reacted with the T cell surface as judged by indirect immunofluorescence, and by its mitogenicity for quiescent peripheral blood lymphocytes. Immunohistological staining and immunoprecipitation experiments showed that both the T3‐delta and T3‐epsilon chains are T cell‐specific. As seen with the anti‐alpha/beta chain reagent WT‐31, anti‐T3‐delta chain monoclonal antibodies stained medullary thymocytes more intensely than cortical thymocytes, whereas the difference between the staining of cortical and medullary thymocytes was generally not apparent with anti‐T3‐epsilon chain antibodies. Because of this specificity and their ability to react with both the denatured and the native forms of each polypeptide chain, these new monoclonal reagents will be useful tools in studies of the biosynthesis and cell surface expression of the T3/T cell receptor complex during normal and malignant thymic differentiation.

During intrathymic differentiation, the genes coding for the T cell receptor/CD3 (TCR/CD3) complex are expressed in stages resulting in surface expression of competent receptors on the most mature T cells. The CD3‐delta, gamma and epsilon proteins, which are expressed intracellularly in the earliest detectable cells of T lineage, form the core of the TCR/CD3 complex. In the present investigation aimed at understanding the tissue specific expression of the murine CD3‐delta gene, a T cell specific DNase I hypersensitive site was found 0.6 kb downstream of the polyadenylation site of the gene. A 0.4 kb genomic fragment encompassing this hypersensitive site exhibited properties of a strictly T cell‐specific transcriptional enhancer which acts in a position‐ and orientation‐independent manner. However, the activity of the promoter region was not found to be T cell restricted. Within this novel T cell‐specific enhancer region, sequence motifs were found which are shared with the promoters of both the mouse and human CD3‐delta genes; in this region we also found sequence homologies to the enhancer core element of the thymotropic viruses SL3 and RadLV. These findings suggest that regulatory elements 3′ of the CD3‐delta gene are at least in part responsible for its exclusive expression in thymus‐derived lymphocytes.

Amino-acid and carbohydrate compositions in addition to limited NH2-terminal sequence data of four preparations of HLA antigens, including products of both of the major histocompatibility loci (HLA-A and HLA-B), show that the antigens have great homology in the primary structure. A striking observation is that HLA antigens also have a considerable structure homology with mouse H-2 antigens. HLA antigens appear to have one carbohydrate side chain which has the same composition in all four cases. The possible relation of the structure of the heavy chain of HLA antigens to immunoglobulins is discussed, based on the limited data which are available so far.

The T3 complex is composed of three polypeptide chains that are both structurally and functionally associated with the receptor for antigen on the surface of human T lymphocytes. In a series of experiments utilizing both somatic cell hybrids and chromosomal hybridization in situ, the genes encoding two members of the human T3 complex, T3-delta and T3-epsilon, were found to reside on the long arm of chromosome 11 in band q23. The murine T3-epsilon gene was localized to chromosome 9. The location of the T3-delta and T3-epsilon genes with respect to the Hu-ets-1 gene, which is also located in 11q23, is discussed. Recent assignments of several genes, preferentially expressed in human cells of hematopoietic and neuroectodermal origins, to band q23 of human chromosome 11 and the murine equivalents to murine chromosome 9 may define a conserved gene cluster important in cell proliferation and differentiation.

The antigen receptor of the T lymphocyte consists of two variable T-cell receptor chains (either TCR-alpha, TCR-beta or TCR-gamma, TCR-delta) noncovalently linked to four different invariant membrane proteins (CD3-gamma, CD3-delta, CD3-epsilon, and the CD3-zeta homodimer). The CD3 genes are expressed early in thymocyte development, preceding the rearrangement and expression of the T-cell receptor genes. Here we report the isolation and structural analysis of the human CD3-epsilon gene. The gene consisted of nine exons. Three exons, encoding the junction of leader peptide and mature protein, were extremely small (21, 15, and 18 base pairs, respectively). The murine gene contained only two such miniexons, the sequences of which were not homologous to those of the three human miniexons. But from comparisons of intron sequences the regions surrounding the human miniexons III and IV appeared to be closely related to those surrounding the murine miniexons III and IV. The most-3' miniexon in the human gene (IVa) had no murine counterpart and appeared not to duplicate any of the other miniexons. Sequence analysis of CD3-epsilon cDNA clones isolated from four independent libraries gave no evidence for alternative use of these miniexons. Like CD3-delta, the CD3-epsilon gene was transcribed from a weak, nontissue-specific, TATA-less promoter. Pulsed-field electrophoresis showed that the human CD3-epsilon gene was separated from the CD3-gamma, CD3-delta gene pair by at least 30 kilobases, but by no more than 300 kilobases.

The T cell antigen receptor (TCR) consists of a disulfide-linked TCR-alpha/beta heterodimer that is both structurally and functionally associated with a set of four non-covalently linked membrane proteins termed CD3-gamma, -delta, -epsilon, and -zeta. An additional protein described recently, CD3-omega, has been suggested to play a role in assembly of the CD3 complex on the basis of its transient association with the CD3 proteins early during biosynthesis. Association of all the proteins seems to be a prerequisite for intracellular transport, since mutants lacking either the TCR-alpha or -beta protein do not express the CD3 complex on the cell surface. CD3-cDNAs were transfected into COS cells in order to study the protein-protein interactions ruling the assembly of the CD3 macromolecular structure. CD3-delta-epsilon, CD3-gamma-epsilon, and CD3-gamma-delta-epsilon intermediates could be detected. These data indicated that a CD3 core structure could be formed in the absence of the other members of the complex (CD3-zeta, -omega, TCR-alpha, and -beta). Both the individual CD3 chains and the assembled CD3.gamma.delta.epsilon complexes could not be detected on the cellular surface but in an intracellular compartment, probably the endoplasmic reticulum or the cis Golgi. The transfection experiments allowed us to identify the 25-kDa member of the murine CD3 complex as CD3-epsilon m. Furthermore, a 23-kDa glycoprotein seen upon metabolic labeling of human T cells was shown to be an immature form of the CD3-gamma h protein.

Background & Aims: We describe a type of colitis that develops after transplantation of nonallogeneic wt bone marrow cells into T cell- and natural killer cell-deficient Tg@e26 mice (BM->Tg@e26). In these animals, severe wasting and inflammation of the colon correlates with the expansion of mucosal T lymphocytes that displays cytotoxic activity. The aims of this study were to determine the relative contribution of perforin and Fas ligand (Fas-L) expression to the cytotoxic action of these T cells and to examine the influence of each pathway in this model of colitis. Methods: Colonic T cells were tested for their ability to mediate Fas- and perforin-dependent killing in redirected cytotoxicity assays. Bone marrow cells from donor mice lacking either Fas-L (gld mice) or perforin (PFP^n^u^l^l mice) or both molecules were used to reconstitute Tg@e26 mice. Results: Colon cytotoxic T lymphocyte displayed both Fas- and perforin-dependent killing. Deficiency in perforin, but not Fas-L, resulted in reduced incidence of wasting and, to a lesser extent, severe colitis in BM->Tg@e26 animals. Conclusions: Colon T cells from BM->Tg@e26 mice express both perforin and Fas-L. Although neither pathway is critical in the development of colitis, perforin does have a measurable influence on disease in the BM->Tg@e26 colitis model. GASTROENTEROLOGY 1998;115:849-855

Activation-induced cell death (AICD) occurs primarily in recently activated T cells after a second TCR triggering. Since a threshold in the activation status may be critical for AICD, it is likely that the CD3 ITAM, docking sites for tyrosine kinases, regulate AICD. A 'threshold model' for AICD was tested by using two targeted mutant mouse strains lacking either the ζ chain (CD3ζ^-/-) or the ITAM of the ζ chain (CD3ζ^-/-:Tg^ζΔ67-150). Although the T cells from the CD3ζ^-/- mice express extremely low levels of surface TCR, a subpopulation ( 18%) of activated T cells could be induced to express TCR/FcεRI γ by using a powerful polyclonal activation protocol. These activated TCR/FcεRI γ T cells were capable of undergoing AICD, but its induction required 10 times as much anti-CD3ε mAb as that required for AICD of wild-type T cells. Thus, the intensity of AICD correlated with the level of CD3 expression and was less efficient with activated, CD3ζ^-/--derived T cells. By contrast, AICD of T cells from the CD3ζ^-/-:Tg^αΔ67-150 mice could be induced with low doses of anti-CD3ε mAb and the extent of AICD was comparable to T cells from wild-type mice. The AICD induced in T cells from CD3ζ^-/-, CD3ζ^-/-:Tg^ζΔ67-150 and normal controls was specifically inhibited by Fas-Ig fusion proteins. Our data support the 'threshold model' of AICD by demonstrating that AICD is controlled by the strength of T cell activation.
Keywords:activation-induced cell death, apoptosis, CD3ζ^-/-, CD3ζ^-/-:Tg^ζΔ67-150, death signal, Fcγ receptor

A novel approach for identification of adenine and guanine nucleotide-binding proteins in permeabilized cells is described. Cells were incubated for various periods with α-32P-labeled nucleotides and then briefly treated with periodate. Condensation products formed in situbetween the protein bound α-32P-labeled oxidized nucleotide (NTPoxi) and a lysine residue near the nucleotide-binding sites were rapidly stabilized by the addition of cyanoborohydride. Analysis by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that in the human leukemic T-cell line Jurkat a number of distinct intracellular proteins could be labeled with ATPoxi(Mr40,000-200,000) or GTPoxi(Mr19,000-80,000). Competition with deoxyribonucleotides confirmed the selectivity of these affinity labeling reactions. To test this method two classical GTP-binding proteins were further examined. First the α-subunits of the Gsand Gi−2proteins were specifically labeled with [α-32P]-GTPoxibut not with [α-32P]ATPoxi. Second, p21raswas crosslinked specifically to [α-32P]GTPoxior to its bound endogenous ligand. Surprisingly, under optimized conditions 60% of the rasprotein was specifically modified, demonstrating the high efficiency and sensitivity of the method. As a first step toward isolation of hitherto unidentified nucleotide-binding proteins, rabbit antisera specific for the modified amino acid residues were raised. The presented labeling method can be applied for identification of nucleotide-binding proteins in all eukaryotic cells.