A transgenic mouse model to analyze CD8+ effector T cell differentiation in vivo

Antigen-specific effector T cells are prerequisite to immune protection, but because of the lack of effector cell-specific markers, their generation and differentiation has been difficult to study. We report that effector cells are highly enriched in a T cell subset that can be specifically identified in transgenic (T-GFP) mice expressing green fluorescent protein (GFP) under control of the murine CD4 promoter and proximal enhancer. Consistent with previous studies of these transcriptional control elements, GFP was strongly and specifically expressed in nearly all resting and short-term activated CD4+ and CD8+ T cells. However, when T-GFP mice were challenged with vaccinia virus, allogeneic tumor cells, or staphylococcal enterotoxin A, the cytotoxic and IFN-γ-producing T cells lost GFP expression. Upon T cell receptor (TCR) ligation by αCD3, sorted GFP+ cells fluxed calcium and proliferated vigorously. In contrast, GFP− effector cells showed a diminished calcium flux and did not proliferate. Instead, they underwent apoptosis unless supplied with exogenous IL-2. By reverse transcription–PCR analysis, the GFP− cells up-regulated the pro-apoptotic molecule, Fas-L, and down-regulated gene expression of the proximal TCR signaling molecule, CD3ζ, and c-jun, a component of the AP-1 transcription factor. Thus, differential regulation of TCR signaling may explain the divergent responses of naïve and effector T cells to antigen stimulation.

Functional analysis of DNA bending and unwinding by the high mobility group domain of LEF-1

LEF-1 (lymphoid enhancer-binding factor 1) is a cell type-specific member of the family of high mobility group (HMG) domain proteins that recognizes a specific nucleotide sequence in the T cell receptor (TCR) α enhancer. In this study, we extend the analysis of the DNA-binding properties of LEF-1 and examine their contributions to the regulation of gene expression. We find that LEF-1, like nonspecific HMG-domain proteins, can interact with irregular DNA structures such as four-way junctions, albeit with lower efficiency than with specific duplex DNA. We also show by a phasing analysis that the LEF-induced DNA bend is directed toward the major groove. In addition, we find that the interaction of LEF-1 with a specific binding site in circular DNA changes the linking number of DNA and unwinds the double helix. Finally, we identified two nucleotides in the LEF-1-binding site that are important for protein-induced DNA bending. Mutations of these nucleotides decrease both the extent of DNA bending and the transactivation of the TCRα enhancer by LEF-1, suggesting a contribution of protein-induced DNA bending to the function of TCRα enhancer.

Inhibition of intrathymic T cell development by expression of a transgenic antagonist peptide

The mature T cell receptor (TCR) repertoire is shaped by positive- and negative-selection events taking place during T cell development. These events are regulated by interactions between the TCR and major histocompatibility complex molecules presenting self-peptides. It has been shown that many antagonist peptides are efficient at mediating positive selection. In this study we analyzed the effects of a transgene encoding an antagonist peptide (influenza NP34) that is presented by H-2Db in a Tap-1-independent fashion in mice expressing the influenza NP68-specific TCR F5. We find that the transgenic peptide does not mediate positive or negative selection in F5+Tap-1−/− mice, but inhibits maturation of CD8+ single positive thymocytes in F5+Tap-1+ mice without inducing signs of negative selection. We conclude that antagonism of antigen recognition occurs not only at the level of mature T cells but also in T cell development.

Perturbation of the T cell repertoire in rheumatoid arthritis

Aberrations in the T cell repertoire with the emergence of oligoclonal populations have been described in patients with rheumatoid arthritis (RA). However, the extent of the repertoire perturbations as well as the underlying mechanisms are not known. We now have examined the diversity of the peripheral CD4 T cell repertoire by determining the frequencies of arbitrarily selected T cell receptor (TCR) β-chain sequences. Healthy individuals displayed a highly diverse repertoire, with a median frequency of individual TCR β-chain sequences of 1 in 2.4 × 107 CD4 T cells. In RA patients, the median TCR β-chain frequency was increased 10-fold, indicating marked contraction of the repertoire (P < 0.001). The loss in TCR diversity was not limited to CD4 memory T cells but also involved the compartment of naive T cells, suggesting that it reflected an abnormality in T cell repertoire formation and not a consequence of antigen recognition in the synovium. Also, control patients with chronic inflammatory disease such as hepatitis C expressed a diverse repertoire indistinguishable from that of normals. Telomere length studies indicated an increased replicative history of peripheral CD4 T cells in RA patients, suggesting an enhanced turnover within the CD4 compartment. Compared with age-matched controls, terminal restriction fragment sizes were 1.7 kilobases shorter (P < 0.001). These data demonstrate an altered CD4 T cell homeostasis in RA that may contribute to the autoimmune response as well as to the immunodeficiency in these patients.

Identification of an early T cell progenitor for a pathway of T cell maturation in the bone marrow

We have identified a rare (≈0.05–0.1%) population of cells (Thy-1hiCD16+CD44hiCD2−TCRαβ−B220−Mac-1−NK1.1−) in the adult mouse bone marrow that generates CD4+ and CD8+ TCRαβ+ T cells after tissue culture for 48 hr in the presence of Ly5 congenic marrow cells. The essential stages in the maturation of the progenitors were determined; the stages included an early transition from CD2−CD16+CD44hiTCRαβ− to CD2+CD16int/−CD44int/−TCRαβ− cells, and a later transition to CD4+CD8+TCRαβ+ double-positive T cells that rapidly generate the CD4+ and CD8+ single-positive T cells. The maturation of the progenitors is almost completely arrested at the CD2+TCRαβ− stage by the presence of mature T cells at the initiation of cultures. This alternate pathway is supported by the marrow microenvironment; it recapitulates critical intermediary steps in intrathymic T cell maturation.

Specific requirement for CD3ɛ in T cell development

T cell antigen receptor (TCR) and pre-TCR complexes are composed of clonotypic heterodimers in association with dimers of signal transducing invariant subunits (CD3γ, -δ, -ɛ, and ζ). The role of individual invariant subunits in T cell development has been investigated by generating gene-specific mutations in mice. Mutation of CD3γ, -δ, or ζ results in an incomplete block in development, characterized by reduced numbers of mature T cells that express low levels of TCR. In contrast, mature T cells are absent from CD3ɛ−/− mice, and thymocyte development is arrested at the early CD4−CD8− stage. Although these results suggest that CD3ɛ is essential for pre-TCR and TCR expression/function, their interpretation is complicated by the fact that expression of the CD3γ and CD3δ genes also is reduced in CD3ɛ−/− mice. Thus, it is unclear whether the phenotype of CD3ɛ−/− mice reflects the collective effects of CD3γ, -δ, and -ɛ deficiency. By removing the selectable marker (PGK-NEO) from the targeted CD3ɛ gene via Cre/loxP-mediated recombination, we generated mice that lack CD3ɛ yet retain normal expression of the closely linked CD3γ and CD3δ genes. These (CD3ɛΔ/Δ) mice exhibited an early arrest in T cell development, similar to that of CD3ɛ−/− mice. Moreover, the developmental defect could be rescued by expression of a CD3ɛ transgene. These results identify an essential role for CD3ɛ in T cell development not shared by the CD3γ, CD3δ, or ζ-family proteins and provide further evidence that PGK-NEO can influence the expression of genes in its proximity.

Jun NH2-terminal kinase is constitutively activated in T cells transformed by the intracellular parasite Theileria parva

When T cells become infected by the parasite Theileria parva, they acquire a transformed phenotype and no longer require antigen-specific stimulation or exogenous growth factors. This is accompanied by constitutive interleukin 2 (IL-2) and IL-2 receptor expression. Transformation can be reversed entirely by elimination of the parasites using the specific drug BW720c. Extracellular signal-regulated kinase and jun NH2-terminal kinase (JNK) are members of the mitogen-activated protein kinase family, which play a central role in the regulation of cellular differentiation and proliferation and also participate in the regulation of IL-2 and IL-2 receptor gene expression. T. parva was found to induce an unorthodox pattern of mitogen-activated protein kinase expression in infected T cells. JNK-1 and JNK-2 are constitutively active in a parasite-dependent manner, but have altered properties. In contrast, extracellular signal-regulated kinase-2 is not activated even though its activation pathway is functionally intact. Different components of the T cell receptor (TCR)-dependent signal transduction pathways also were examined. The TCRζ or CD3ɛ chains were found not to be phosphorylated and T. parva-transformed T cells were resistant to inhibitors that block the early steps of T cell activation. Compounds that inhibit the progression of T cells to proliferation, however, were inhibitory. Our data provide the first example, to our knowledge, for parasite-mediated JNK activation, and our findings strongly suggest that T. parva not only lifts the requirement for antigenic stimulation but also entirely bypasses early TCR-dependent signal transduction pathways to induce continuous proliferation.

An enhancer-blocking element between α and δ gene segments within the human T cell receptor α/δ locus

T cell receptor (TCR) α and δ gene segments are organized within a single genetic locus but are differentially regulated during T cell development. An enhancer-blocking element (BEAD-1, for blocking element alpha/delta 1) was localized to a 2.0-kb region 3′ of TCR δ gene segments and 5′ of TCR α joining gene segments within this locus. BEAD-1 blocked the ability of the TCR δ enhancer (Eδ) to activate a promoter when located between the two in a chromatin-integrated construct. We propose that BEAD-1 functions as a boundary that separates the TCR α/δ locus into distinct regulatory domains controlled by Eδ and the TCR α enhancer, and that it prevents Eδ from opening the chromatin of the TCR α joining gene segments for VDJ recombination at an early stage of T cell development.

A novel multigene family encodes diversified variable regions

Antigen recognition in the adaptive immune response by Ig and T-cell antigen receptors (TCRs) is effected through patterned differences in the peptide sequence in the V regions. V-region specificity forms through genetically programmed rearrangement of individual, diversified segmental elements in single somatic cells. Other Ig superfamily members, including natural killer receptors that mediate cell-surface recognition, do not undergo segmental reorganization, and contain type-2 C (C2) domains, which are structurally distinct from the C1 domains found in Ig and TCR. Immunoreceptor tyrosine-based inhibitory motifs that transduce negative regulatory signals through the cell membrane are found in certain natural killer and other cell surface inhibitory receptors, but not in Ig and TCR. In this study, we employ a genomic approach by using the pufferfish (Spheroides nephelus) to characterize a nonrearranging novel immune-type receptor gene family. Twenty-six different nonrearranging genes, which each encode highly diversified V as well as a V-like C2 extracellular domain, a transmembrane region, and in most instances, an immunoreceptor tyrosine-based inhibitory motif-containing cytoplasmic tail, are identified in an ≈113 kb P1 artificial chromosome insert. The presence in novel immune-type receptor genes of V regions that are related closely to those found in Ig and TCR as well as regulatory motifs that are characteristic of inhibitory receptors implies a heretofore unrecognized link between known receptors that mediate adaptive and innate immune functions.

A model for spontaneous B-lineage lymphomas in IgHμ-HOX11 transgenic mice

HOX11, a divergent homeodomain-containing transcription factor, was isolated from the breakpoint of the nonrandom t(10;14)(q24;q11) chromosome translocation found in human T cell acute lymphoblastic leukemias. The translocation places the HOX11 coding sequence under the transcriptional control of TCR α/δ regulatory elements, resulting in ectopic expression of a normal HOX11 protein in thymocytes. To investigate the oncogenic potential of HOX11, we targeted its expression in lymphocytes of transgenic mice by placing the human cellular DNA under the transcriptional control of Ig heavy chain or LCK regulatory sequences. Only IgHμ-HOX11 mice expressing low levels of HOX11 were viable. During their second year of life, all HOX11 transgenic mice became terminally ill with more than 75% developing large cell lymphomas in the spleen, which frequently disseminated to thymus, lymph nodes, and other nonhematopoietic tissues. Lymphoma cells were predominantly clonal IgM+IgD+ mature B cells. Repopulation of severe combined immunodeficient mice with cells from hyperplastic spleens indicated that the HOX11 tumor phenotype was transplantable. Before tumor development, expression of the transgene did not result in perturbations in lymphopoiesis; however, lymphoid hyperplasia involving the splenic marginal zones was present in 20% of spleens. Our studies provide direct evidence that expression of HOX11 in lymphocytes leads to malignant transformation. These mice are a useful model system to study mechanisms involved in transformation from B-lineage hyperplasia to malignant lymphoma and for testing novel approaches to therapy. They represent a novel animal model for non-Hodgkin’s lymphoma of peripheral mature B cell origin.

The ATPase Domain but Not the Acidic Region of Cockayne Syndrome Group B Gene Product Is Essential for DNA Repair

Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA and CSB, are required for transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes certain lesions rapidly and efficiently from the transcribed strand of active genes. CS proteins have also been implicated in the recovery of transcription after certain types of DNA damage such as those lesions induced by UV light. In this study, site-directed mutations have been introduced to the human CSB gene to investigate the functional significance of the conserved ATPase domain and of a highly acidic region of the protein. The CSB mutant alleles were tested for genetic complementation of UV-sensitive phenotypes in the human CS-B homologue of hamster UV61. In addition, the CSB mutant alleles were tested for their ability to complement the sensitivity of UV61 cells to the carcinogen 4-nitroquinoline-1-oxide (4-NQO), which introduces bulky DNA adducts repaired by global genome repair. Point mutation of a highly conserved glutamic acid residue in ATPase motif II abolished the ability of CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery, and gene-specific repair. These data indicate that the integrity of the ATPase domain is critical for CSB function in vivo. Likewise, the CSB ATPase point mutant failed to confer cellular resistance to 4-NQO, suggesting that ATP hydrolysis is required for CSB function in a TCR-independent pathway. On the contrary, a large deletion of the acidic region of CSB protein did not impair the genetic function in the processing of either UV- or 4-NQO-induced DNA damage. Thus the acidic region of CSB is likely to be dispensable for DNA repair, whereas the ATPase domain is essential for CSB function in both TCR-dependent and -independent pathways.

In vitro evolution of a T cell receptor with high affinity for peptide/MHC

T cell receptors (TCRs) exhibit genetic and structural diversity similar to antibodies, but they have binding affinities that are several orders of magnitude lower. It has been suggested that TCRs undergo selection in vivo to maintain lower affinities. Here, we show that there is not an inherent genetic or structural limitation on higher affinity. Higher-affinity TCR variants were generated in the absence of in vivo selective pressures by using yeast display and selection from a library of Vα CDR3 mutants. Selected mutants had greater than 100-fold higher affinity (KD ≈ 9 nM) for the peptide/MHC ligand while retaining a high degree of peptide specificity. Among the high-affinity TCR mutants, a strong preference was found for CDR3α that contained Pro or Gly residues. Finally, unlike the wild-type TCR, a soluble monomeric form of a high-affinity TCR was capable of directly detecting peptide/MHC complexes on antigen-presenting cells. These findings prove that affinity maturation of TCRs is possible and suggest a strategy for engineering TCRs that can be used in targeting specific peptide/MHC complexes for diagnostic and therapeutic purposes.

Transfected Drosophila cells as a probe for defining the minimal requirements for stimulating unprimed CD8+ T cells

Stimulation of naive T cells by antigen-presenting cells (APC) is thought to involve two qualitatively different signals: signal one results from T-cell receptor (TCR) recognition of antigenic peptides bound to major histocompatibility complex (MHC) molecules, whereas signal two reflects contact with one or more costimulatory molecules. The requirements for stimulating naive T cells were studied with MHC class I-restricted CD8+ T cells from a T-cell receptor transgenic line, with defined peptides as antigen and transfected Drosophila cells as APC. Three main findings are reported. First, stimulation of naive T cells via signal one alone (MHC plus peptide) was essentially nonimmunogenic; thus T cells cultured with peptides presented by MHC class I-transfected Drosophila APC lacking costimulatory molecules showed little or no change in their surface phenotype. Second, cotransfection of two costimulatory molecules, B7-1 and intercellular adhesion molecule 1 (ICAM-1), converted class I+ Drosophila cells to potent APC capable of inducing strong T-proliferative responses and cytokine (interleukin 2) production. Third, B7-1 and ICAM-1 acted synergistically, indicating that signal two is complex; synergy between B7-1 and ICAM-1 varied from moderate to extreme and was influenced by both the dose and affinity of the peptide used and the parameter of T-cell activation studied. Transfected Drosophila cells are thus a useful tool for examining the minimal APC requirements for naive T cells.

Tuning of activation thresholds explains flexibility in the selection and development of T cells in the thymus

Immature CD4+CD8+ thymocytes expressing T-cell antigen receptors (TCR) are selected by TCR-mediated recognition of peptides associated with major histocompatibility complex molecules on thymic stromal cells. Selection ensures reactivity of the mature cells to foreign antigens and tolerance to self. Although much has been learned about the factors that determine whether a thymocyte with a given specificity will be positively or negatively selected, selection as an aspect of the developmental process as a whole is less well-understood. Here we invoke a model in which thymocytes tune their response characteristics individually and dynamically in the course of development. Cellular development and selection are driven by receptor-mediated metabolic perturbations. Perturbation is a measure of the net intracellular change induced by external stimulation. It results from the integration of several signals and countersignals over time and therefore depends on the environment and the maturation stage of the cell. Individual cell adaptation limits the range of perturbations. Such adaptation renders thymocytes less sensitive to the level of stimulation per se, but responsive to environmental changes in that level. This formulation begins to explain the mechanisms that link developmental and selection events to each other.

Complementary mutations in an antigenic peptide allow for crossreactivity of autoreactive T-cell clones

T cells recognize antigen by formation of a trimolecular complex in which the T-cell receptor (TCR) recognizes a specific peptide antigen within the groove of a major histocompatibility complex (MHC) molecule. It has generally been assumed that T-cell recognition of two distinct MHC–antigen complexes is due to similarities in the three-dimensional structure of the complexes. Here we report results of experiments examining the crossreactivity of TCRs recognizing the myelin basic protein peptide MBPp85–99 and several of its analogs in the context of MHC. We demonstrate that single conservative amino acid substitutions of the antigenic peptide at the predominant TCR contact residues at positions 91 and 93 totally abrogate reactivity of specific T-cell clones. Yet, when a conservative substitution is made at position 91 concomitant with a substitution at position 93, the T-cell clones regain reactivity equivalent with that of the original stimulating peptide. Thus, the exact nature of the amino acid side chains engaging one TCR functional pocket may change the apparent selectivity of the other predominant TCR functional pocket, thus suggesting a remarkable degree of receptor plasticity. This ability of the TCR–MHC–peptide complex to undergo conformational changes provides a conceptual framework for reconciling the apparent paradox of the extreme selectivity of the TCR and its remarkable crossreactivity with different MHC–peptide complexes.