TCRα reporter mice reveal contribution of dual TCRα expression to T cell repertoire and function.

It is known that a subpopulation of T cells expresses two T cell receptor (TCR) clonotypes, though the extent and functional significance of this is not established. To definitively evaluate dual TCRα cells, we generated mice with green fluorescent protein and red fluorescent protein reporters linked to TCRα, revealing that ∼16% of T cells express dual TCRs, notably higher than prior estimates. Importantly, dual TCR expression has functional consequences, as dual TCR cells predominated response to lymphocytic choriomeningitis virus infection, comprising up to 60% of virus-specific CD4+ and CD8+ T cells during acute responses. Dual receptor expression selectively influenced immune memory, as postinfection memory CD4+ populations contained significantly increased frequencies of dual TCR cells. These data reveal a previously unappreciated contribution of dual TCR cells to the immune repertoire and highlight their potential effects on immune responses.

Generation of Novel Traj18-Deficient Mice Lacking Vα14 Natural Killer T Cells with an Undisturbed T Cell Receptor α-Chain Repertoire.

Invariant Vα14 natural killer T (NKT) cells, characterized by the expression of a single invariant T cell receptor (TCR) α chain encoded by rearranged Trav11 (Vα14)-Traj18 (Jα18) gene segments in mice, and TRAV10 (Vα24)-TRAJ18 (Jα18) in humans, mediate adjuvant effects to activate various effector cell types in both innate and adaptive immune systems that facilitates the potent antitumor effects. It was recently reported that the Jα18-deficient mouse described by our group in 1997 harbors perturbed TCRα repertoire, which raised concerns regarding the validity of some of the experimental conclusions that have been made using this mouse line. To resolve this concern, we generated a novel Traj18-deficient mouse line by specifically targeting the Traj18 gene segment using Cre-Lox approach. Here we showed the newly generated Traj18-deficient mouse has, apart from the absence of Traj18, an undisturbed TCRα chain repertoire by using next generation sequencing and by detecting normal generation of Vα19Jα33 expressing mucosal associated invariant T cells, whose development was abrogated in the originally described Jα18-KO mice. We also demonstrated here the definitive requirement for NKT cells in the protection against tumors and their potent adjuvant effects on antigen-specific CD8 T cells.

Chitin microparticles for the control of intestinal inflammation.

BACKGROUND: Chitin is a polymer of N-acetylglucosamine with the ability to regulate innate and adaptive immune responses. However, the detailed mechanisms of chitin-mediated regulation of intestinal inflammation are only partially known. METHODS: In this study chitin microparticles (CMPs) or phosphate-buffered saline (PBS) were orally administered to acute and chronic colitis models every 3 days for 6 consecutive weeks beginning at weaning age. The effects of this treatment were evaluated by histology, cytokine production, coculture study, and enteric bacterial analysis in dextran sodium sulfate (DSS)-induced colitis or T-cell receptor alpha (TCRα) knockout chronic colitis models. RESULTS: Histologically, chitin-treated mice showed significantly suppressed colitis as compared with PBS-treated mice in both animal models. The production of interferon-gamma (IFN-γ) was upregulated in the mucosa of chitin-treated mice compared with control mice. The major source of IFN-γ-producing cells was CD4+ T cells. In mouse dendritic cells (DCs) we found that CMPs were efficiently internalized and processed within 48 hours. Mesenteric lymph nodes (MLNs) CD4+ T cells isolated from chitin-treated mice produced a 7-fold higher amount of IFN-γ in the culture supernatant after being cocultured with DCs and chitin as compared with the control. Proliferation of carboxyfluorescein succinimidyl ester (CFSE)(low) CD4+ T cells in MLNs and enteric bacterial translocation rates were significantly reduced in chitin-treated mice when compared with the control. In addition, CMPs improved the imbalance of enteric bacterial compositions and significantly increased interleukin (IL)-10-producing cells in noninflamed colon, indicating the immunoregulatory effects of CMPs in intestinal mucosa. CONCLUSIONS: CMPs significantly suppress the development of inflammation by modulating cytokine balance and microbial environment in colon.

SEL1L, the homologue of yeast Hrd3p, is involved in protein dislocation from the mammalian ER.

Protein quality control in the endoplasmic reticulum (ER) involves recognition of misfolded proteins and dislocation from the ER lumen into the cytosol, followed by proteasomal degradation. Viruses have co-opted this pathway to destroy proteins that are crucial for host defense. Examination of dislocation of class I major histocompatibility complex (MHC) heavy chains (HCs) catalyzed by the human cytomegalovirus (HCMV) immunoevasin US11 uncovered a conserved complex of the mammalian dislocation machinery. We analyze the contributions of a novel complex member, SEL1L, mammalian homologue of yHrd3p, to the dislocation process. Perturbation of SEL1L function discriminates between the dislocation pathways used by US11 and US2, which is a second HCMV protein that catalyzes dislocation of class I MHC HCs. Furthermore, reduction of the level of SEL1L by small hairpin RNA (shRNA) inhibits the degradation of a misfolded ribophorin fragment (RI332) independently of the presence of viral accessories. These results allow us to place SEL1L in the broader context of glycoprotein degradation, and imply the existence of multiple independent modes of extraction of misfolded substrates from the mammalian ER.

Staphylococcal enterotoxin H induces V alpha-specific expansion of T cells.

Staphylococcal enterotoxin H (SEH) is a bacterial superantigen secreted by Staphylococcus aureus. Superantigens are presented on the MHC class II and activate large amounts of T cells by cross-linking APC and T cells. In this study, RT-PCR was used to show that SEH stimulates human T cells via the Valpha domain of TCR, in particular Valpha10 (TRAV27), while no TCR Vbeta-specific expansion was seen. This is in sharp contrast to all other studied bacterial superantigens, which are highly specific for TCR Vbeta. It was further confirmed by flow cytometry that SEH stimulation does not alter the levels of certain TCR Vbeta. In a functional assay addressing cross-reactivity, Vbeta binding superantigens were found to form one group, whereas SEH has different properties that fit well with Valpha reactivity. As SEH binds on top of MHC class II, an interaction between MHC and TCR upon SEH binding is not likely. This concludes that the specific expansion of TCR Valpha is not due to contacts between MHC and TCR, instead we suggest that SEH directly interacts with the TCR Valpha domain.

T cell receptor (TCR) alpha/delta locus enhancer identity and position are critical for the assembly of TCR delta and alpha variable region genes.

T cell receptor (TCR) delta and alpha variable region genes are assembled from germ-line gene segments located in a single chromosomal locus in which TCR delta segments are situated between TCR alpha segments. The TCR alpha enhancer (E alpha) located at the 3' end of the TCR alpha/delta locus functions over a long chromosomal distance to promote TCR alpha rearrangement and maximal TCR delta expression; whereas the TCR delta enhancer (E delta) is located among the TCR delta segments and functions with additional element(s) to mediate TCR delta rearrangement. We used gene-targeted mutation to evaluate whether the identity of E alpha and the position of E delta are critical for the developmental stage-specific assembly of TCR delta and alpha variable region genes. Specific replacement of E alpha with E delta, the core E alpha element (E alpha C), or the Ig heavy chain intronic enhancer (iE mu), all of which promote accessibility in the context of transgenic V(D)J recombination substrates, did not promote a significant level of TCR alpha rearrangement beyond that observed in the absence of E alpha. Therefore, the identity and full complement of E alpha-binding sites are critical for promoting accessibility within the TCR alpha locus. In the absence of the endogenous E delta element, specific replacement of E alpha with E delta also did not promote TCR delta rearrangement. However, deletion of intervening TCR alpha/delta locus sequences to restore the inserted E delta to its normal chromosomal position relative to 5' sequences rescued TCR delta rearrangement. Therefore, unlike E alpha, E delta lacks ability to function over the large intervening TCR alpha locus and or E delta function requires proximity to additional upstream element(s) to promote TCR delta accessibility.

Human invariant V alpha 24-J alpha Q TCR supports the development of CD1d-dependent NK1.1+ and NK1.1- T cells in transgenic mice.

A sizable fraction of T cells expressing the NK cell marker NK1.1 (NKT cells) bear a very conserved TCR, characterized by homologous invariant (inv.) TCR V alpha 24-J alpha Q and V alpha 14-J alpha 18 rearrangements in humans and mice, respectively, and are thus defined as inv. NKT cells. Because human inv. NKT cells recognize mouse CD1d in vitro, we wondered whether a human inv. V alpha 24 TCR could be selected in vivo by mouse ligands presented by CD1d, thereby supporting the development of inv. NKT cells in mice. Therefore, we generated transgenic (Tg) mice expressing the human inv. V alpha 24-J alpha Q TCR chain in all T cells. The expression of the human inv. V alpha 24 TCR in TCR C alpha(-/-) mice indeed rescues the development of inv. NKT cells, which home preferentially to the liver and respond to the CD1d-restricted ligand alpha-galactosylceramide (alpha-GalCer). However, unlike inv. NKT cells from non-Tg mice, the majority of NKT cells in V alpha 24 Tg mice display a double-negative phenotype, as well as a significant increase in TCR V beta 7 and a corresponding decrease in TCR V beta 8.2 use. Despite the forced expression of the human CD1d-restricted TCR in C alpha(-/-) mice, staining with mCD1d-alpha-GalCer tetramers reveals that the absolute numbers of peripheral CD1d-dependent T lymphocytes increase at most by 2-fold. This increase is accounted for mainly by an increased fraction of NK1.1(-) T cells that bind CD1d-alpha-GalCer tetramers. These findings indicate that human inv. V alpha 24 TCR supports the development of CD1d-dependent lymphocytes in mice, and argue for a tight homeostatic control on the total number of inv. NKT cells. Thus, human inv. V alpha 24 TCR-expressing mice are a valuable model to study different aspects of the inv. NKT cell subset.

Evaluation of in vitro and in vivo activity of benzindazole-4,9-quinones against Cryptosporidium parvum.

A series of benzindazole-4,9-quinones was tested for growth-inhibitory effects on Cryptosporidium parvum in vitro and in vivo. Most compounds showed considerable activity at concentrations from 25 to 100 micro M. For instance, at 25 micro M the derivatives 5-hydroxy-8-chloro-N1-methylbenz[f]-indazole-4,9-quinone and 5-chloro-N2-methylbenz[f]indazole-4,9-quinone inhibited growth of C. parvum 78-100%, and at 50 micro M seven of the 23 derivatives inhibited growth > or = 90%. The activity of the former two compounds was confirmed in a T-cell receptor alpha (TCR-alpha)-deficient mouse model of chronic cryptosporidiosis. In these mice, the mean infectivity scores (IS) in the caecum were 0.63-0.20, whereas in sham-treated mice the score was 1.44 (P < 0.05). There were similar differences in IS in the ileum, where the score for treated mice was 1.12-0.20 and that for mice receiving no drug was 1.32. There was no acute or chronic toxicity for any compound tested in vivo.

A role for the alpha-chain connecting peptide motif in mediating TCR-CD8 cooperation.

To generate peripheral T cells that are both self-MHC restricted and self-MHC tolerant, thymocytes are subjected to positive and negative selection. How the TCR discriminates between positive and negative selection ligands is not well understood, although there is substantial evidence that the CD4 and CD8 coreceptors play an important role in this cell fate decision. We have previously identified an evolutionarily conserved motif in the TCR, the alpha-chain connecting peptide motif (alpha-CPM), which allows the TCR to deliver positive selection signals. Thymocytes expressing alpha-CPM-deficient receptors do not undergo positive selection, whereas their negative selection is not impaired. In this work we studied the ligand binding and receptor function of alpha-CPM-deficient TCRs by generating T cell hybridomas expressing wild-type or alpha-CPM-deficient forms of the T1 TCR. This K(d)-restricted TCR is specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide(252-260) IASA-YIPSAEK(ABA)I and is therefore amenable to TCR photoaffinity labeling. The experiments presented in this work show that alpha-CPM-deficient TCRs fail to cooperate with CD8 to enhance ligand binding and functional responses.

Premature TCR alpha beta expression and signaling in early thymocytes impair thymocyte expansion and partially block their development.

In thymocyte ontogeny, Tcr-a genes rearrange after Tcr-b genes. TCR alpha beta transgenic (Tg) mice have no such delay, consequently expressing rearranged TCR alpha beta proteins early in the ontogeny. Such mice exhibit reduced thymic cellularity and accumulate mature, nonprecursor TCR(+)CD8(-)4(-) thymocytes, believed to be caused by premature Tg TCR alpha beta expression via unknown mechanism(s). Here, we show that premature expression of TCR alpha beta on early thymocytes curtails thymocyte expansion and impairs the CD8(-)4(-) --> CD8(+)4(+) transition. This effect is accomplished by two distinct mechanisms. First, the early formation of TCR alpha beta appears to impair the formation and function of pre-TCR, consistent with recently published results. Second, the premature TCR alpha beta contact with intrathymic MHC molecules further pronounces the block in proliferation and differentiation. These results suggest that the benefit of asynchronous Tcr-a and Tcr-b rearrangement is not only to minimize waste during thymopoiesis, but also to simultaneously allow proper expression/function of the pre-TCR and to shield CD8(-)4(-) thymocytes from TCR alpha beta signals that impair thymocyte proliferation and CD8(-)4(-) --> CD8(+)4(+) transition.

Competitive displacement of pT alpha by TCR-alpha during TCR assembly prevents surface coexpression of pre-TCR and alpha beta TCR.

During alphabeta T cell development, CD4(-)CD8(-) thymocytes first express pre-TCR (pTalpha/TCR-beta) before their differentiation to the CD4(+)CD8(+) stage. Positive selection of self-tolerant T cells is then determined by the alphabeta TCR expressed on CD4(+)CD8(+) thymocytes. Conceivably, an overlap in surface expression of these two receptors would interfere with the delicate balance of thymic selection. Therefore, a mechanism ensuring the sequential expression of pre-TCR and TCR must function during thymocyte development. In support of this notion, we demonstrate that expression of TCR-alpha by immature thymocytes terminates the surface expression of pre-TCR. Our results reveal that expression of TCR-alpha precludes the formation of pTalpha/TCR-beta dimers within the endoplasmic reticulum, leading to the displacement of pre-TCR from the cell surface. These findings illustrate a novel posttranslational mechanism for the regulation of pre-TCR expression, which may ensure that alphabeta TCR expression on thymocytes undergoing selection is not compromised by the expression of pre-TCR.

Control of organ-specific demethylation by an element of the T-cell receptor-alpha locus control region.

DNA methylation is important for mammalian development and the control of gene expression. Recent data suggest that DNA methylation causes chromatin closure and gene silencing. During development, tissue specifically expressed gene loci become selectively demethylated in the appropriate cell types by poorly understood processes. Locus control regions (LCRs), which are cis-acting elements providing stable, tissue-specific expression to linked transgenes in chromatin, may play a role in tissue-specific DNA demethylation. We studied the methylation status of the LCR for the mouse T-cell receptor alpha/delta locus using a novel assay for scanning large distances of DNA for methylation sites. Tissue-specific functions of this LCR depend largely on two DNase I-hypersensitive site clusters (HS), HS1 (T-cell receptor alpha enhancer) and HS1'. We report that these HS induce lymphoid organ-specific DNA demethylation in a region located 3.8 kilobases away with little effect on intervening, methylated DNA. This demethylation is impaired in mice with a germline deletion of the HS1/HS1' clusters. Using 5'-deletion mutants of a transgenic LCR reporter gene construct, we show that HS1' can act in the absence of HS1 to direct this tissue-specific DNA demethylation event. Thus, elements of an LCR can control tissue-specific DNA methylation patterns both in transgenes and inside its native locus.