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T cells recognize antigens through the interaction of their T cell receptor (TCR) with a peptide-major histocompatibility complex (pMHC) molecule. Following thymic-positive selection, TCRs in peripheral naive T cells are expected to bind MHC alleles of the host. Peripheral clonal selection is expected to further increase the frequency of antigen-specific TCRs that bind to the host MHC alleles. To check for a systematic preference for MHC-binding T cells in TCR repertoires, we developed Natural Language Processing-based methods to predict TCR–MHC binding independently of the peptide presented for Class I MHC alleles. We trained a classifier on published TCR–pMHC binding pairs and obtained a high area under curve (AUC) of over 0.90 on the test set. However, when applied to TCR repertoires, the accuracy of the classifier dropped. We thus developed a two-stage prediction model, based on large-scale naive and memory TCR repertoires, denoted TCR HLA-binding predictor (CLAIRE). Since each host carries multiple human leukocyte antigen (HLA) alleles, we first computed whether a TCR on a CD8 T cell binds an MHC from any of the host Class-I HLA alleles. We then performed an iteration, where we predict the binding with the most probable allele from the first round. We show that this classifier is more precise for memory than for naïve cells. Moreover, it can be transferred between datasets. Finally, we developed a CD4–CD8 T cell classifier to apply CLAIRE to unsorted bulk sequencing datasets and showed a high AUC of 0.96 and 0.90 on large datasets. CLAIRE is available through a GitHub at: https://github.com/louzounlab/CLAIRE, and as a server at: https://claire.math.biu.ac.il/Home. [ABSTRACT FROM AUTHOR]

Signaling pathways in innate and adaptive immunity play vital roles in pathogen recognition and the functions of immune cells. Higher-order assemblies have recently emerged as a central principle that governs immune signaling and, by extension, cellular communication in general. There are mainly two types of higher-order assemblies: 1) ordered, solid-like large supramolecular complexes formed by stable and rigid protein-protein interactions, and 2) liquid-like phase-separated condensates formed by weaker and more dynamic intermolecular interactions. This review covers key examples of both types of higher-order assemblies in major immune pathways. By placing emphasis on the molecular structures of the examples provided, we discuss how their structural organization enables elegant mechanisms of signaling regulation. [ABSTRACT FROM AUTHOR]

Summary: Renal cell carcinoma (RCC) is comprised of clear‐cell (ccRCC) and non‐clear‐cell (nccRCC) tumors. Despite definitive surgical resection in localized disease, recurrence often occurs. A commercial method based on a multiplex polymerase chain reaction (PCR) assay exclusively targets rearranged T cell receptor (TCR) genes to generate high‐throughput sequencing‐based data, allowing characterization of the immune repertoire within tumors. In this study we performed a retrospective analysis on archived tumor samples from patients with recurring versus non‐recurring T3 ccRCC and on samples from early nccRCC versus ccRCC. Following genomic DNA extraction and multiplex PCR, the fraction of T cells within tumors, the number of unique receptors ('richness') and their relative abundances ('clonality') were calculated. Statistical significance and correlations were calculated using Student's t‐test and Spearman's rho, respectively. Average fraction and clonality of T cells in tumors from non‐recurring patients was 2.5‐ and 4.3‐fold higher than in recurring patients (P = 0.025 and P = 0.043, respectively). A significant positive correlation was found between T cell fraction and clonality (Spearman's rho = 0.78, P = 0.008). The average fraction of T cells in ccRCC tumors was 2.8‐fold higher than in nccRCC tumors (P = 0.015). Clonality and estimated richness were similar between ccRCC and nccRCC tumors. In summary, recurrence of ccRCC is associated with a lower fraction and clonality of T cells within tumors; nccRCC tumors are more 'deserted' than ccRCC, but similar in their ability to generate a clonal T cell repertoire. Our work suggests associations between the characteristics of T cell infiltrate, histology and tumor recurrence. [ABSTRACT FROM AUTHOR]

Metabolic regulation has been proven to play a critical role in T cell antitumor immunity. However, cholesterol metabolism as a key component of this regulation remains largely unexplored. Herein, we found that the low-density lipoprotein receptor (LDLR), which has been previously identified as a transporter for cholesterol, plays a pivotal role in regulating CD8+ T cell antitumor activity. Besides the involvement of cholesterol uptake which is mediated by LDLR in T cell priming and clonal expansion, we also found a non-canonical function of LDLR in CD8+ T cells: LDLR interacts with the T-cell receptor (TCR) complex and regulates TCR recycling and signaling, thus facilitating the effector function of cytotoxic T-lymphocytes (CTLs). Furthermore, we found that the tumor microenvironment (TME) downregulates CD8+ T cell LDLR level and TCR signaling via tumor cell-derived proprotein convertase subtilisin/kexin type 9 (PCSK9) which binds to LDLR and prevents the recycling of LDLR and TCR to the plasma membrane thus inhibits the effector function of CTLs. Moreover, genetic deletion or pharmacological inhibition of PCSK9 in tumor cells can enhance the antitumor activity of CD8+ T cells by alleviating the suppressive effect on CD8+ T cells and consequently inhibit tumor progression. While previously established as a hypercholesterolemia target, this study highlights PCSK9/LDLR as a potential target for cancer immunotherapy as well. [ABSTRACT FROM AUTHOR]

The neonatal period presents a complex scenario where the threshold of reactivity toward colonizing microbiota, maternal antigens, autoantigens, and pathogens must be carefully moderated and balanced. CD8+ T cells are critical for the response against intracellular bacteria and viruses, but this immune compartment maintains altered function relative to adult counterparts because of the unique challenges which infants face. Here, we review our current understanding of the factors which may promote the attenuation and altered function of the neonatal CD8+ T‐cell response and potential avenues for future study. Specifically, we have focused on the neonatal CD8+ T‐cell ontogeny, memory formation, TCR structure and repertoire, TCR inhibitory receptors, and the clinical implications of altered neonatal CD8+ T‐cell function. Special emphasis has been placed on examining the response of preterm neonates relative to term neonates and adults. [ABSTRACT FROM AUTHOR]

The limited application of immunotherapy in acute myeloid leukemia (AML) may be due to poor understanding of the global T cell immune dysfunction in AML. In this study, we analyzed the distribution characteristics of 24 TCR Vβ subfamilies in CD3+, CD4+, and CD8+ T cells in AML patients and healthy controls. The percentage of TCR Vβ subfamily T cells was predominately lower in most AML cases, while it was increased in some cases. TCR Vβ2+T cells were increased in AML, particularly TCR Vβ2+CD4+T cells, which were significantly higher. To further address the immunosuppression in different Vβ subfamilies, we characterized the distribution of program death‐1 (PD‐1)+T cells in TCR Vβ subfamilies of CD4+ and CD8+T cells. Significantly higher levels of PD‐1+Vβ+T cells were found for most Vβ subfamilies in most AML cases. A higher percentage of PD‐1+Vβ2+T cells with a high number of Vβ2+T cells was found in all of the CD3+, CD4+, and CD8+ T cell subsets. Moreover, increasing PD‐1+Vβ7.2, Vβ8+, Vβ14+, Vβ16+, and Vβ22+CD8+T cells were distributed in the AML‐M5 subtype group compared with the AML‐M3 group. In addition, higher PD‐1+ Vβ5.2+ and PD‐1+ Vβ12+CD8+T cells were associated with AML patients who had a poor response to chemotherapy. In conclusion, increased PD‐1+Vβ+T cells is a common characteristic of AML, higher PD‐1+Vβ2+T cells may be associated with a low antileukemia effect, and higher PD‐1+Vβ5.2+ and PD‐1+Vβ12+CD8+T cells may be related to poor prognosis in AML. These characteristics may be worth considering as immune biomarkers for clinical outcome in AML. [ABSTRACT FROM AUTHOR]

Summary: The antigenic specificity of T cells occurs via generation and rearrangement of different gene segments producing a functional T cell receptor (TCR). High‐throughput sequencing (HTS) allows in‐depth assessment of TCR repertoire patterns. There are limited data concerning whether TCR repertoires are altered in inflammatory bowel disease. We hypothesized that pediatric ulcerative colitis (UC) patients possess unique TCR repertoires, resulting from clonotypical expansions in the gut. Paired blood and rectal samples were collected from nine newly diagnosed treatment‐naive pediatric UC patients and four healthy controls. DNA was isolated to determine the TCR‐β repertoire by HTS. Significant clonal expansion was demonstrated in UC patients, with inverse correlation between clinical disease severity and repertoire diversity in the gut. Using different repertoire variables in rectal biopsies, a clear segregation was observed between patients with severe UC, those with mild–moderate disease and healthy controls. Moreover, the overlap between autologous blood–rectal samples in UC patients was significantly higher compared with overlap among controls. Finally, we identified several clonotypes that were shared in either all or the majority of UC patients in the colon. Clonal expansion of TCR‐β‐expressing T cells among UC patients correlates with disease severity and highlights their involvement in mediating intestinal inflammation. Using next‐generation sequencing of the TCR‐β we demonstrate altered repertoire features in pediatric patients with newly diagnosed ulcerative colitis. We show clonal expansion of specific clonotypes that correlates with disease activity in ulcerative colitis, highlighting their involvement in mediating intestinal inflammation. [ABSTRACT FROM AUTHOR]

Approximately 10% of peripheral T cells express 2 functional TCR αβ heterodimers. Receptor co‐expression changes the repertoire of TCRs produced during thymic development, enabling generation of T cells bearing TCRs not capable of mediating positive selection or that would normally be negatively selected. The effect of receptor co‐expression on the composition and functionality of the peripheral TCR repertoire is not well defined, though evidence demonstrates dual TCR cells pose an increased risk for unwanted immune responses such as autoimmunity and alloreactivity. Based on our previous finding that dual TCR expression promotes positive selection, we hypothesized that dual TCR expression may enhance T cell homeostasis via increased reactivity against self‐peptide:MHC (pMHC) ligands. To examine the effect of dual TCR expression on T cell homeostasis, we performed cotransfer experiments comparing T cells genetically deficient for dual TCR expression (TCRα+/−) with wild‐type T cells in models of acute and chronic lymphopenia‐induced proliferation (LIP). Lack of dual TCR expression resulted in reduced LIP. The effect of dual TCR expression on LIP was most pronounced in acute lymphopenia, which is driven by recognition of low‐affinity self‐pMHC ligands. Differences in homeostatic proliferation were not attributable to differences in total TCR expression or signaling, but were dependent on interaction with MHC and associated with increased affinity for positively selecting self‐pMHC as evidenced by higher expression of CD5 by dual TCR cells from wild‐type mice. These results represent an unappreciated novel mechanism driving homeostasis and shaping the T cell repertoire, potentially promoting autoreactive or heterologous immune responses. Co‐expression of 2 TCRs enhances homeostatic proliferation in settings of acute lymphopenia, driving post‐transplant expansion of dual TCR cells with known auto‐ and alloreactivity. [ABSTRACT FROM AUTHOR]

Certain food components possess immunomodulatory effects. The aim of this study was to elucidate the mechanism of the immunostimulatory activity of Brassica rapa L. We demonstrated an enhancement of natural killer (NK) activity and interferon (IFN)-γ production in mice that were orally administered an insoluble fraction of B. rapa L. The insoluble fraction of B. rapa L. significantly induced IFN-γ production in mouse spleen cells in an interleukin (IL)-12-dependent manner, and NK1.1+ cells were the main cells responsible for producing IFN-γ. Additionally, the results suggested that the active compounds in the insoluble fraction were recognized by Toll-like receptor (TLR) 2, TLR4, and C-type lectin receptors on dendritic cells, and they activated signaling cascades such as MAPK, NF-κB, and Syk. These findings suggest that B. rapa L. is a potentially promising immuno-improving material, and it might be useful for preventing immunological disorders such as infections and cancers by activating innate immunity. [ABSTRACT FROM AUTHOR]

Background and Aim: T cells are key players in the chronic intestinal inflammation that characterises Crohn's disease. Here we aim to map the intestinal T-cell receptor [TCR] repertoire in patients with Crohn's disease, using next-generation sequencing technology to examine the clonality of the T-cell compartment in relation to mucosal inflammation and response to therapy. Methods: Biopsies were taken from endoscopically inflamed and uninflamed ileum and colon of 19 patients with Crohn's disease. From this cohort, additional biopsies were taken after 8 weeks of remission induction therapy from eight responders and eight non-responders. Control biopsies from 11 patients without inflammatory bowel disease [IBD] were included. The TCRβ repertoire was analysed by next-generation sequencing of biopsy RNA. Results: Both in Crohn's disease patients and in non-IBD controls, a broad intestinal T-cell repertoire was found, with a considerable part consisting of expanded clones. Clones in Crohn's disease were more expanded [p = 0.008], with the largest clones representing up to as much as 58% of the total repertoire. There was a substantial overlap of the repertoire between inflamed and uninflamed tissue and between ileum and colon. Following therapy, responders showed larger changes in the T-cell repertoire than non-responders, although a considerable part of the repertoire remained unchanged in both groups. Conclusions: The intestinal T-cell repertoire distribution in Crohn's disease is different from that in the normal gut, containing profoundly expanded T-cell clones that take up a large part of the repertoire. The T-cell repertoire is fairly stable regardless of endoscopic mucosal inflammation or response to therapy. [ABSTRACT FROM AUTHOR]

Polyspecific T cells recognizing multiple distinct self-antigens have been identified in multiple sclerosis and other organ-specific autoimmune diseases, but their pathophysiological relevance remains undetermined. Using a mouse model of multiple sclerosis, we show that autoimmune encephalomyelitis induction is strictly dependent on reactivation of pathogenic T cells by a peptide (35-55) derived from myelin oligodendrocyte glycoprotein (MOG). This disease-inducing response wanes after onset. Strikingly, the progression of disease is driven by the in situ activation and expansion of a minority of MOG35-55-specific T cells that also recognize neurofilament-medium (NF-M)15-35, an intermediate filament protein expressed in neurons. This mobilization of bispecific T cells is critical for disease progression as adoptive transfer of NF-M15-35/MOG35-55 bispecific T cell lines caused full-blown disease in wild-type but not NF-M-deficient recipients. Moreover, specific tolerance through injection of NF-M15-35 peptide at the peak of disease halted experimental autoimmune encephalomyelitis progression. Our findings highlight the importance of polyspecific autoreactive T cells in the aggravation and perpetuation of central nervous system autoimmunity. [ABSTRACT FROM AUTHOR]

CD4+ T helper lymphocytes (Th) orchestrate the immune response after their activation by antigenpresenting cells. Activation of naïve Th cells is reported to generate the reduction in surface epitopes of sialic acid (Sia) in α2,3 and α2,6 linkages. In this work, we report that in spite of this glycophenotype, anti-CD3/anti-CD28-activated purified human naïve Th cells show a significant increase in surface Sia, as assessed by metabolic labeling, compared with resting naïve Th cells, suggesting an increased flux of Sia toward Siaa2,8 glycoconjugates. To understand this increase as a result of ganglioside up-regulation, we observed that very early after activation, human naïve Th cells show an increased expression in surface GD3 and neoexpression of surface GD2 gangliosides, the latter clustering with the T cell receptor (TCR). Also, we report that in contrast to GM2/GD2 synthase null mice, lentiviral vector-mediated silencing of the GM2/GD2 synthase in activated human naïve Th cells reduced efficient TCR clustering and downstream signaling, as assessed by proliferation assays and IL-2 and IL-2R expression, pointing to an important role of this enzyme in activation of human naive Th cells. [ABSTRACT FROM AUTHOR]

Emerging data indicate that particular major histocompatibility complex (MHC)-bound antigenic peptides can be recognized by identical or near-identical αβ T cell receptors (TCRs) in different individuals. To establish the functional relevance of this phenomenon, we artificially paired α and β chains from closely related TCRs specific for the human leucocyte antigen (HLA)-B*35:01-restricted HIV-1 negative regulatory factor (Nef)-derived epitope VY8 (VPLRPMTY, residues 74-81). Several hybrid TCRs generated in this manner failed to express at the cell surface, despite near homology with naturally isolated αβ chain combinations. Moreover, a substantial proportion of those αβ TCRs that did express lost specificity for the index VY8 peptide sequence. One such hybrid αβ pair gained neo-variant specificity in the context of the VY8 backbone. Collectively, these data show that clonotypically similar TCRs can display profound differences in surface expression, antigen specificity and cross-reactivity with potential relevance for the control of mutable viruses. [ABSTRACT FROM AUTHOR]

The murine immune system is not necessarily identical to it human counterpart, which has led to the construction of humanized mice. The current study analysed whether or not a human immune system contained within the non-obese diabetic ( NOD)- Rag1null-γ chainnull ( NRG) mouse model was an accurate representation of the original stem cell donor and if multiple mice constructed from the same donor were similar to one another. To that end, lightly irradiated NRG mice were injected intrahepatically on day 1 of life with purified cord blood-derived CD34+ stem and progenitor cells. Multiple mice were constructed from each cord blood donor. Mice were analysed quarterly for changes in the immune system, and followed for periods up to 12 months post-transplant. Mice from the same donor were compared directly with each other as well as with the original donor. Analyses were performed for immune reconstitution, including flow cytometry, T cell receptor ( TCR) and B cell receptor ( BCR) spectratyping. It was observed that NRG mice could be 'humanized' long-term using cord blood stem cells, and that animals constructed from the same cord blood donor were nearly identical to one another, but quite different from the original stem cell donor immune system. [ABSTRACT FROM AUTHOR]

Human infection with Trypanosoma cruzi leads to Chagas disease, which presents as several different clinical conditions ranging from an asymptomatic form to a severe dilated cardiomyopathy. Several studies have demonstrated that T cells play a critical role in the development of cardiac pathology, as well as in immunoregulation during chronic disease. However, the mechanisms that drive protective or pathogenic T cell response are not known. We have shown that CD4+ T cells from chagasic patients preferentially express T cell receptor (TCR) β-chain variable region (Vβ) 5. The aim of this work was to determine whether T cells expressing this particular Vβ region displayed variable or restricted CDR3 sequences, as an indicator of the nature of the stimulus leading to the activation of these T cells in vivo. Additionally, we aimed to evaluate phenotypic characteristics of these cells that might be associated with pathology. CDR3 junctional region sequencing of Vβ5·1 expressing CD4+ T cells revealed the occurrence of a highly homologous CDR3 region with conserved TCR Jβ region usage among patients with cardiac, but not indeterminate, Chagas disease. Moreover, correlation analysis indicated that the frequency of CD4+Vβ5·1+ cells is associated with granzyme A expression, suggesting that these cells might display cytotoxic function. Together these results provide new insight into T cell recognition of antigens involved in Chagas disease and suggest that these cells may be implicated in the pathogenesis of chagasic cardiomyopathy. [ABSTRACT FROM AUTHOR]

Membrane microdomains consisting of sphingomyelin (SM) and cholesterol appear to be important for signal transduction in T-cell activation. The present study was designed to elucidate the role of membrane SM in vivo and in vitro using sphingomyelin synthase 1 (SMS1) knock out (SMS1−/−) mice and Concanavalin A (ConA)-induced hepatitis. After establishing SMS1−/− mice, we investigated CD4+ T-cell functions including proliferation, cytokine production and signal transduction in vivo. We also examined severity of hepatitis, cytokine production in serum and liver after ConA injection at a dose of 20 mg kg−1. CD4+ T cells from SMS1−/− mice showed severe deficiency of membrane SM and several profound defects compared with wild-type controls as follows: (i) cellular proliferation and production of IL-2 and IFN-γ by co-cross-linking of CD3 and CD4; (ii) tyrosine phosphorylation of LAT and its association with ZAP-70; (iii) clustering and co-localization of TCR with lipid rafts. Consistent with these impaired CD4+ T-cell functions in vitro, SMS1−/− mice showed decreased serum levels of IL-6 and IFN-γ by ConA injection, which renders SMS1−/− mice less sensitive to ConA-induced hepatitis. These results indicated that the deficiency of membrane SM caused the CD4+ T-cell dysfunction through impaired lipid raft function contributed to protection of ConA-induced liver injury, suggesting that the membrane SM is critical for full T-cell activation both in vitro and in vivo. [ABSTRACT FROM PUBLISHER]

Although mature T cells divide and differentiate when they receive strong TCR stimulation, most immature CD4+CD8+ thymocytes die. The molecular basis for this marked difference in response is not known. Observations that TCR-stimulated CD4+CD8+ thymocytes fail to polarize their microtubule-organizing center (MTOC), one of the first events that occurs upon antigen activation of mature T cells, suggests that TCR signaling routes in immature and mature T cells diverge early and upstream of MTOC polarization. To better understand the source of the divergence, we examined the molecular basis for the difference in TCR-mediated MTOC polarization. We show that unstable microtubules are a feature of immature murine CD4+CD8+ thymocytes, which also exhibit higher levels of glycogen synthase kinase 3 (GSK3) activity, a known inhibitor of microtubule stability. Importantly, CD4+CD8+ thymocytes gained the ability to polarize their MTOC in response to TCR signals when GSK3 activity was inhibited. GSK3 inhibition also abrogated TCR-mediated apoptosis of immature thymocytes. Together, our results suggest that a developmentally regulated difference in GSK3 activity has a major influence on immature CD4+CD8+ thymocyte versus mature T-cell responses to TCR stimulation. [ABSTRACT FROM AUTHOR]

Structural information on how the TCR transmits signals upon binding of its antigen peptide MHC molecule ligand is still lacking. The ectodomains of the TCRα/β, CD3ɛγ and CD3ɛδ dimers, as well as the transmembrane domain of CD3ζ, have been characterized by X-ray crystallography and nuclear magnetic resonance (NMR). However, no structural data have been obtained for the entire TCR complex. In this study, we have purified the TCR from T cells under native conditions and used electron microscopy to derive a three-dimensional structure. The TCR complex appears as a pear-shaped structure of 180 × 120 × 65 . Furthermore, the use of mAbs has allowed to determine the orientation of the TCRα/β and CD3 subunits and to suggest a model of interactions. Interestingly, the reconstructed TCR is larger than expected for a complex with a αβγɛδɛζζ stoichiometry. The accommodation of a second TCRαβ to fill in the extra volume is discussed. [ABSTRACT FROM AUTHOR]

NKT cells are characterized by their production of both Th1 and Th2 cytokines immediately after stimulation with α-galactosylceramide (α-GalCer), which is composed of α-galactopyranose linked to ceramide (itself composed of sphingosine and fatty-acyl chains); the chain length of the ceramide varies and this affects the ability of α-GalCer to stimulate cytokine production. However, the contribution of its galactopyranose sugar moiety remains unclear. We synthesized α-carba-GalCer, which has an α-linked carba-galactosyl moiety; here, the 5a′-oxygen atom of the D-galactopyranose ring of α-GalCer is replaced by a methylene group. The α-carba-GalCer was more stable and showed higher affinity to the NKT receptor. It thus enhanced and prolonged production of IL-12 and IFN-γ compared with α-GalCer, resulting in augmented NKT cell-mediated adjuvant effects in vivo. The α-carba-GalCer, which has an ether linkage, was more resistant to degradation by liver microsomes than was α-GalCer, which has an acetal bond. Modulation of the sugar moiety in glycolipids might therefore provide optimal therapeutic reagents for protective immune responses against tumor or pathogens. [ABSTRACT FROM PUBLISHER]

The crystal structure analysis demonstrated that the hydrophobic amino acid residue (isolecuine/leucine/valine) at conserved position 97 of Vδ2 TCR plays an important role in recognizing the non-peptide antigen. But its importance to protein antigen remains unclear until now. In the present study, we focus on the role of hydrophobic amino acid residue at conserved position 97 of Vδ2 TCR in complementarity determining region (CDR)3δ-mediated binding to protein antigen. We employed CDR3δ peptide and membrane-engineered γδ TCR as detecting molecules with mutated 97 hydrophobic amino acid residue in CDR3δ (nominated as OT10), a Vδ2 CDR3 sequence derived from tumor infiltrating lymphocytes in ovarian epithelial carcinoma (OEC). Binding assays revealed that OT10 peptide and membrane-engineered γδ TCR (γδ TCR transfected cells with OT10 sequence) could bind specifically ovarian tumor cell line (SKOV3). The mutant analysis indicated that any amino acid substitution at position δI97 could abolish the response of the transfected cells to iso-butylamine, a known non-peptide antigen of γδ T cells. But amino acid substitution of isoleucine at position δ97 did not change the responsiveness of γδ TCR transfected cell to protein antigen. Our data suggested that a mechanism other than non-peptide antigen might mediate the recognition of Vδ2γδ T cells for protein antigen. This finding may provide a possibility that γδ TCR recognize different ligands in diversity manners. [ABSTRACT FROM PUBLISHER]

Sustained extracellular signal-regulated kinase (ERK)-signaling plays a critical role in T-cell-mediated IL-2 production. Although many downstream targets are known for ERK, details remain unknown about which molecules play functional roles in IL-2 production. Here, we addressed this question using proteomic analysis of nuclear proteins from TCR-activated T cells and identified hnRNP-K as one of the ERK targets essential for IL-2 production. hnRNP-K was previously shown by others to be a direct substrate of ERK and form complexes with multiple signaling proteins as well as DNA and RNA. Our data showed a clear ERK-dependent increase in one form of hnRNP-K after TCR stimulation. Small interfering RNA-mediated gene knockdown of hnRNP-K expression abrogated IL-2 production by T cells. Moreover, reduction of hnRNP-K expression caused a notable increase in proteolysis of Vav1, a binding target of hnRNP-K. Since Vav1 is an essential molecule for T-cell activation, the data suggest that ERK signaling is required for T-cell activation partly by inhibiting activation-induced proteolysis of Vav1. [ABSTRACT FROM PUBLISHER]

The aim of this study was to find conserved motifs in specific T cell receptor (TCR) α- and β-chains, and to analyse the association between complementarity determining region 3 (CDR3) spectratype and systemic lupus erythematosus (SLE) activity. TCR α-and β-chain CDR3 spectratypes were analysed in 20 SLE patients. The CDR3 spectratypes of three patients were monitored over time, and the CDR3 regions of clonally expanded T cells were sequenced. CDR3 spectratype analysis showed prominent usage of TCR AV8, AV14, AV23, AV30, AV31, BV2, BV8, BV11, BV14, BV16, BV19 and BV24 families in SLE patients. The CDR3 spectratype showed dynamic change correlating with SLE activity. The sequence of the CDR3 region in clonally expanded T cells suggested a conserved GGX amino acid motif in both α- and β-chains. The Ja34 and Jb2s1 region genes were found in high frequency. Both TCR Vα and Vβ gene usage is highly restricted in SLE, suggesting that the TCRs recognize a limited number of antigenic epitopes. The conserved motifs and limited use of joining region genes may indicate the recognition of similar antigenic epitopes in multiple individuals. [ABSTRACT FROM AUTHOR]

Bam32 (B lymphocyte adapter molecule of 32 kDa) is an adapter protein expressed in some hematopoietic cells including B and T lymphocytes. It was previously shown that Bam32-deficient mice have defects in various aspects of B cell activation including B cell receptor (BCR)-induced Erk activation, BCR-induced proliferation and T-independent antibody responses. In this study, we have examined the role of Bam32 in T cell activation using Bam32-deficient mice. By comparing CD4+ T cells from lymph nodes of wild-type and Bam32-deficient mice, we found that Bam32 was required for optimal TCR-induced Erk activation, cytokine production, proliferation and actin-mediated spreading of CD4+ T cells. These results indicate a novel pathway to Erk activation in T cells involving the adapter protein Bam32. [ABSTRACT FROM PUBLISHER]

The recent development of T cell receptor phage display opens up the possibility of engineering human T cell receptors with antibody-like binding properties for cell-surface peptide antigens. In this review we briefly discuss recent developments in molecular targeting of peptide antigens. We then discuss potential clinical applications of engineered high-affinity T cell receptors in autoimmunity and cancer. [ABSTRACT FROM AUTHOR]

Adoptive antigen-specific immunotherapy is an attractive concept for the treatment of cancer because it does not require immunocompetence of patients, and the specificity of transferred lymphocytes can be targeted against tumour-associated antigens that are poorly immunogenic and thus fail to effectively trigger autologous T cell responses. As the isolation andin vitroexpansion of antigen-specific lymphocytes is difficult,‘conventional’ adoptive T cell therapy can only be carried out in specialized centres in small numbers of patients. However, T cell receptor (TCR) genes isolated from antigen-specific T cells can be exploited as generic therapeutic molecules for‘unconventional’ antigen-specific immunotherapy. Retroviral TCR gene transfer into patient T cells can readily produce populations of antigen-specific lymphocytes after a single round of polyclonal T cell stimulation. TCR gene modified lymphocytes are functionally competentin vitro, and can have therapeutic efficacy in murine modelsin vivo. TCR gene expression is stable and modified lymphocytes can develop into memory T cells. Introduction of TCR genes into CD8+ and CD4+ lymphocytes provides an opportunity to use the same TCR specificity to produce antigen-specific killer and helper T lymphocytes. Thus, TCR gene therapy provides an attractive strategy to develop antigen-specific immunotherapy with autologous lymphocytes as a generic treatment option. [ABSTRACT FROM AUTHOR]

Provides information about bacterial superantigens. Presence of enterotoxic properties in bacterial superantigens; Types of bacterial superantigens.

TCR signaling is mediated by intracellular signaling molecules and nuclear transcription factors, which are tightly regulated by interaction with regulatory proteins such as Grb2 and SLAP. We reported recently that TCR stimulation induces the expression of cytokine‐induced SH2 protein (CIS). The expression of CIS promotes TCR‐mediated activation. We have now found specific interactions between CIS and activated protein kinase C (PKC) α, β and ϑ in TCR‐stimulated T cells. CIS was shown by in vitro kinase assay to associate with activated PKC. In CIS‐expressing T cells isolated from CIS‐transgenic mice, the amount of activated PKC associated with CIS was found to increase following TCR stimulation. By immunohistochemical analysis, CIS was also found to co‐localize with PKCϑ at the plasma membrane of activated T cells. In addition to the interaction and intracellular co‐localization of the CIS and PKC, an increase in the activation of AP‐1 and NF‐κB was noted in CIS‐expressing T cells, after stimulation by either anti‐CD3/CD28 or phorbol myristate acetate + ionomycin. These results suggest that CIS regulates PKC activation, and that this may be important for the activation of both the AP‐1 and NF‐κB pathways in TCR signaling. [ABSTRACT FROM PUBLISHER]

T cell recognition of antigenic peptides is thought to occur preferentially in the context of self‐MHC. Here, we have tested the ability of four different Kb–peptide combinations to stimulate self‐ and allo‐restricted CTL responses in three different mouse stains. Responder T cells were primed in vitro with peptide‐loaded stimulator cells, followed by limiting dilution assays to measure the number of peptide‐specific cytotoxic T lymphocytes (CTL). For three peptides the number of CTL restricted by self‐MHC was higher than for allo‐MHC‐restricted responses, although the difference was surprisingly small (3‐ to 5‐fold). For the fourth peptide there was no detectable difference in the number of self‐ and allo‐restricted CTL. Peptide titration experiments revealed that high avidity CTL were present in both the self‐ and allo‐restricted setting. These data showed that the bias for preferred peptide recognition in the context of self‐MHC imposed by positive thymic selection seems marginal. This raised the possibility that the TCR repertoire is inherently biased towards MHC restriction, independent of MHC‐guided thymic selection. This was supported by the analysis of mature T cells generated from the thymus of MHC‐deficient mice by lectin stimulation. Kb‐restricted CTL were found amongst these T cells at numbers similar to those of allo‐restricted CTL. In summary, the data suggest that MHC‐restricted peptide recognition is an inherent feature of the TCR repertoire and does not require thymic selection by MHC molecules. [ABSTRACT FROM PUBLISHER]

SUMMARY T cells are thought to play an important regulatory role in atopic asthma. We hypothesized that human blood and BAL T cell subsets bearing various TCR-Vβ genes might show selective differences in their cytokine profile. Peripheral blood (PB) and bronchoalveolar lavage (BAL) T cells from seven atopic asthmatic and six non-atopic non-asthmatic subjects were stimulated with PMA and ionomycin in the presence of monensin and analysed for TCR-Vβ expression and production of cytokines at the single cell level. The percentage of IFN-γ- and IL-2-producing BAL T cells was elevated compared with PB T cells from both the asthmatic subjects and the non-atopic, non-asthmatic controls. A small percentage of PB and BAL T cells produced IL-4 and IL-5, in asthmatic and normal subjects. In peripheral blood, the percentage of T cells expressing each cytokine was similar in the various TCR-Vβ subsets and in total CD3+ T cells in all normal and six of seven asthmatic subjects. However, there was a substantial degree of heterogeneity in the cytokine profile of BAL TCR-Vβ subsets compared with the total CD3+ T cells. This was more obvious in the asthmatic subjects with a reduction in the percentage of IFN-γ- and IL-2-expressing T cells (five of seven asthmatic subjects) and an increase in the percentage of IL-4- and IL-5-expressing T cells (two of seven asthmatic subjects). These data confirm previous findings of an elevated proportion of IFN-γ- and IL-2-producing BAL T cells while only a small proportion of PB and BAL T cells produce IL-4 and IL-5. Moreover, subsets of BAL T cells, defined by their TCR-Vβ usage, may differ in their cytokine profile compared with the total CD3+ T cells, implying that T cells expressing different Vβ elements may play different roles in regulating the airway inflammation in asthma. [ABSTRACT FROM AUTHOR]

Although there has been extensive analysis on the capacity of MHC–peptide tetramers to bind antigen-specific TCR, there have been comparatively few studies regarding the role of the CD4 and CD8 co-receptors in binding and activation by these multimeric molecules. Here, we start from the observation that different antibodies against human CD8 exert opposite effects on MHC–peptide tetramer binding to the TCR: tetramer staining was enhanced by OKT8 antibody, while it was blocked with SK1 antibody. We used these different anti-CD8 antibodies to modulate CD8 function during tetramer staining of Melan-A/MART1-specific CTL clones. We show that CD8 action could be variably modulated during all the phases of interaction, indicating that CD8 participates in both the initial association of the TCR with MHC–peptide tetramers and the stability of this interaction. While the blocking effect of anti-CD8 antibodies was mostly exerted during the initial binding of the TCR with MHC–peptide tetramers, the enhancing effect was exerted by augmenting the duration of this interaction. Blocking anti-CD8 antibodies were also capable of preventing tetramer-mediated T cell activation. The possibility of variably affecting MHC–peptide tetramer binding and T cell activation using anti-CD8 antibodies confirms the critical role exerted by the CD8 co-receptor in this interaction and supports the notion that TCR engagement by MHC–peptide ligands typically involves CD8. [ABSTRACT FROM PUBLISHER]

Many murine T cell clones grow continuously in culture despite weekly ligation of their TCR by antigen. To learn how the cultured cells avoid or minimize antigen-induced cell death (AICD), we compared Fas and tumor necrosis factor (TNF) receptors (TNFR) on several long-term cultured CD8+ T cell clones with those on naive and activated naive cells expressing the same TCR (2C). In contrast to the naive cells, Fas was absent on the cultured clones and the TNFR-II receptor, present initially at high levels on the cultured cells, was rapidly down-modulated in response to TCR ligation and had virtually disappeared by 2 h, when only ~10% of the cloned cells had been induced to express TNF-α. The extent of AICD of the cultured clones in response to cognate peptide–MHC on the presenting cells used for routine stimulation of the cultures was also considerably less than the massive cell death of the clones following exposure to anti-CD3 antibody plate-bound at high density. [ABSTRACT FROM PUBLISHER]

Clinically distinct forms of childhood arthritis are associated with different risk alleles of polymorphic loci within the MHC, which code for the antigen-presenting class I or class II molecules. We have compared the TCR diversity of synovial T cells from children with enthesitis-related (HLA-B27+) arthritis and oligoarticular arthritis (with class II MHC risk allele associations) in parallel with peripheral blood T cells from each child, using a high-resolution heteroduplex TCR analysis. We demonstrate that multiple clonal T cell expansions are present and persistent within the joint in both groups, but that there is disease-specific divergence in the dominant T cell subset containing these expansions. Thus, the largest clonotypes within the inflamed joints of children with class II-associated arthritis are within the CD4+ synovial T cell population, while the dominant clones from children with enthesitis-related arthritis (associated with a class I allele) are within the CD8+ synovial T cell population. These data provide powerful data to support the concept that recognition of MHC–peptide complexes by T cells plays a role in the pathogenesis of juvenile arthritis. [ABSTRACT FROM PUBLISHER]

We have shown that the dominance of CD8+ T cells expressing TCR Vβ17 in the adult HLA-A*0201-restricted influenza A/M158–66-specific response is acquired following first antigen exposure. Despite the acquired dominance of Vβ17+ cells, subdominant M158–66-specific clones expressing non-Vβ17+ TCR persist in all individuals. To determine whether the affinity of the expressed TCR for the HLA-A*0201/M158–66 complex could influence functional properties, M158–66-specific clones expressing subdominant (non-Vβ17+) TCR were compared to cytotoxic T lymphocyte (CTL) clones expressing dominant (Vβ17+) TCR. The Vβ17+ CTL required up to 10,000-fold lower amounts of M1 peptide to mediate lysis compared to CTL clones expressing other Vβ gene segments. All Vβ17+ CTL clones tested bound HLA-A*0201/M158–66 tetramer, but two of three CTL clones expressing other TCR did not bind tetramer. The inability of non-Vβ17+ CTL to bind tetramer did not correlate with phenotype, CD8 dependence or with cytokine production profiles. This suggests a limitation for the use of tetramers in examining subdominant T cell responses. Together these findings suggest that Vβ17+ CTL which dominate the HLA-A*0201-restricted CTL response against influenza A are not functionally distinct from subdominant non-Vβ17+ CTL. The dominance of Vβ17+ CTL is likely to result from a competitive advantage due to superior CTL avidity for the HLA-A*0201/M158–66 complex. [ABSTRACT FROM PUBLISHER]

During acute human viral infections, such as influenza A, specific cytotoxic T lymphocytes (CTL) are generated which aid virus clearance. We have observed that in HLA-A*0201+ subjects, CTL expressing Vβ17+ TCR and recognizing a peptide from the influenza A matrix protein (M158–66) dominate this response. In experimental models of infection such dominance can be due to inheritance of a restricted T cell repertoire or acquired consequent on expansion of CTL bearing an optimum TCR conformation against the MHC–peptide complex. To examine how influenza A infection might influence the development of TCR V0β17 expansion, we studied influenza A-specific CTL in a cross-sectional study of 82 HLA-A*0201+ individuals from birth (cord blood) to adulthood. Primary M158–66 -specific CTL were detected in cord blood, but their TCR were diverse and depletion of Vβ17+ cells did not abrogate specific cytotoxicity. In contrast following natural influenza A infection, TCR Vβ17+ CTL dominated to the extent that only one of nine adult CTL lines retained any functional activity after in vitro depletion of Vβ17+ CTL. These results suggest that the dominance of Vβ17+ TCR among adult M158–66-specific CTL results from maturation and focussing of the response driven by exposure to influenza, and have implications for optimum immunization strategies. [ABSTRACT FROM PUBLISHER]

In this pilot study, we address the nature of the re-population of the T-cell compartment in HIV-1+ (Human Immunodeficiency Virus 1), vertically infected children placed on successful regimens of HAART (highly active anti-retroviral therapy) incorporating 2 NRTI and a protease inhibitor. The clonality of the T-cell compartment and the abundance of RTEs (Recent Thymic Emigrants) were determined 2 weeks before and 20 weeks after initiation of HAART in a subgroup of children taking part in the PENTA (Paediatric European Network for the Treatment of AIDS) 5 trial. Analysis of the clonality of the circulating T-cell compartment was assessed using CDR3 spectratyping and analysed using the Kolmogorov–Smirnov two sample test. This revealed that a high degree of T-cell clonal restriction still exists 5 months into therapy, despite the appearance of previously undetectable T-cell clones within the periphery. We detected no increase in RTE abundance in this 5 month period, as determined by PCR detection of TRECs (T-Cell Receptor Excision Circles). We conclude that the observed re-population of T cells within the periphery of treated children is heavily reliant upon the maintenance/expansion of pre-existing cells during the 5 month period immediately following the initiation of therapy. [ABSTRACT FROM AUTHOR]

To further define the molecular basis of tolerance to a peripherally expressed antigen we have correlated differences in functional capacity with biochemical events in hemagglutinin (HA)-specific cytotoxic T lymphocyte (CTL) clones derived either from a conventional B10.D2 mouse that is not tolerant to HA (D2 Clone 6) or from an InsHA mouse that is tolerant to HA (InsHA Clone 12). D2 Clone 6, but not InsHA Clone 12, triggers diabetes following in vivo transfer into irradiated InsHA hosts. This diabetogenic clone shows complete and sustained phosphorylation of TCR ζζ chain and ZAP-70 following stimulation with HA-pulsed antigen-presenting cells. In contrast, InsHA Clone 12 showed only partial phosphorylation of TCR ζζ and no phosphorylation of ZAP-70. There was no defect in activation or recruitment of Lck to the TCR complex in both the clones following stimulation with the cognate antigen. This deficiency in the proximal signaling in the InsHA Clone 12 could be overcome by increasing the strength of signal through the CD3–TCR complex, indicating that the signaling machinery of InsHA Clone 12 was functional. These data demonstrate that the HA-responsive CD8+ T cells that can be retrieved from InsHA mice after tolerance induction respond to HA as a partial agonist/antagonist. [ABSTRACT FROM PUBLISHER]

In a recent study we identified certain asymptomatic individuals infected by Ebola virus (EBOV) who mounted specific IgG and early and strong inflammatory responses. Here, we further characterized the primary immune response to EBOV during the course of asymptomatic infection in humans. Inflammatory responses occurred in temporal association with anti-inflammatory phase composed by soluble antagonist IL-1RA, circulating TNF receptors, IL-10 and cortisol. At the end of the inflammatory process, mRNA expression of T-cell cytokines (IL-2 and IL-4) and activation markers (CD28, CD40L and CTLA4) was up-regulated, strongly suggesting T-cell activation. This T-cell activation was followed by EBOV-specific IgG responses (mainly IgG3 ang IgG1), and by marked and sustained up-regulation of IFNγ, FasL and perforin mRNA expression, suggesting activation of cytotoxic cells. The terminal down-regulation of these latter markers coincided with the release of the apoptotic marker 41/7 NMP in blood and with the disappearance of viral RNA from PBMC, suggesting that infected cells are eliminated by cytotoxic mechanisms. Finally, RT-PCR analysis of TCR-Vβ repertoire usage showed that TCR-Vβ12 mRNA was never expressed during the infection. Taken together, these findings improve our understanding about immune response during human asymptomatic Ebola infection, and throw new light on protection against Ebola virus. [ABSTRACT FROM AUTHOR]

DNA vaccination has been used to generate effective cellular as well as humoral immunity against target antigens. Here we have investigated the induction and involvement of regulatory T cell (Treg) responses in mediating prevention of experimental autoimmune encephalomyelitis (EAE), following vaccination with plasmid DNA encoding the TCR Vβ8.2 chain predominantly displayed on disease-causing lymphocytes. Vaccination with DNA encoding the wild-type TCR results in priming of type 1 CD4 Treg and skewing of the global response to myelin basic protein in a Th2 direction, leading to significant protection from disease. In contrast, vaccination with mutant DNA encoding altered residues critically involved in recognition by the Treg results in priming of a type 2 regulatory response which fails to mediate immune deviation or protection from EAE. Control mice immunized with DNA, encoding TCR with changes at an irrelevant site, were protected from antigen-induced disease. Furthermore, protection can be transferred into naive recipients with CD4 Treg from wild-type DNA-immunized mice but not from animals vaccinated with the mutant DNA. These data suggest that vaccination with plasmid DNA encoding one or multiple Vβ genes can be exploited to enhance natural regulatory responses for intervention in autoimmune conditions. [ABSTRACT FROM PUBLISHER]

The efficacy of cytotoxic T lymphocytes (CTL) has been shown to be highly dependent upon their functional avidity (the sensitivity of their cellular response to MHC–peptide complexes). To examine this relationship, we employed target cell lysis as a quantitative measure and established a set of four CTL clones that exhibited a range of functional avidities spanning more than three orders of magnitude. Within this set, clones displayed a linear correlation between functional avidity and the TCR down-regulation that occurred in response to increasing antigen density. Staining intensity of MHC–peptide tetramer, however, correlated only with the very highest and very lowest avidity clones; the two intermediate-avidity clones showed an inverse relationship between tetramer staining and functional avidity. Compensation for differences in surface levels of TCR improved the correlation, but failed to fully account for this discrepancy. Comparison of TCR signals generated by stimulation of CTL with substrate-bound soluble MHC–peptide or antigen-presenting cells suggested that internal TCR signaling efficiency accounts for at least a portion of the observed functional avidity and suggests the need for caution in directly relating tetramer staining to avidity. [ABSTRACT FROM PUBLISHER]

The immunological correlates of highly active antiretroviral therapy (HAART)-induced suppression of human immunodeficiency virus type 1 (HIV-1) replication have been investigated. 20 HIV-1-infected patients with mean CD4+ T cell count of 298/µl, plasma viral load of 4·7 log10 copies/ml and naive for protease inhibitors (PI) were studied during12 months of HAART. An increased number of both CD4+ and CD8+ naive T cells and a normalization of the frequency of CCR5- and CXCR4-expressing CD4+ T cells were readily observed after starting therapy. Single cell analysis of cytokine production after 12 months of HAART showed an increased number of interleukin (IL)-2-, but not IL-4- and (IFN)-γ‐, producing T cells and a decreased percentage of CD8+ IFN-γ + cells. A correlation between the frequency of IFN-γ-producing T cells and that of memory, CCR5+ and CD95+ T cells was demonstrated in both CD4+ and CD8+ subsets. The diversity of T cell receptor (TCR) variable beta (BV) chain repertoire significantly increased after 12 months of HAART within the CD4+ but not the CD8+ T cell subset. However, the level of perturbation of the third complementarity-determining region (CDR3), was not significantly modified by effective therapy. The number of anti-HIV Gag and Pol cytotoxic T lymphocytes precursors (CTLp) decreased during HAART and highly correlated with the CD8 IFN-γ response. Ameliorated clinical conditions were observed in all patients in absence of any opportunistic infections during all the study period. These observations indicate that a better restoration of immunity may be obtained in patients starting HAART at less advanced stages of the disease. [ABSTRACT FROM AUTHOR]

Experimental autoimmune uveoretinitis (EAU), which is a T cell mediated organ specific autoimmune disease, is induced by immunization with interphotoreceptor retinoid binding protein (IRBP) in susceptible strains of mice. It has been found that IRBP-derived peptide 518–529 (p518–529) generates Th2-type responses and inhibits IRBP-induced EAU, indicating that the p518–529 might be an epitope for suppressor T cells in IRBP-induced EAU. First, we observed that there were T cells producing the Th2 type cytokines such as IL-4 and IL-10 in late phase of EAU. Furthermore, to examine whether p518–529-reactive T cells expand in the eye during EAU, T cell receptor (TCR) of ocular T cells was compared with that of p518–529 reactive T cells in spleen from mice with EAU by PCR-single strand conformation polymorphism (PCR-SSCP) and nucleotide sequence analysis. SSCP and sequence analyses indicated that p518–529 reactive TCR BV10+ T cells bearing amino acid motif(PWG) and TCR BV13+ T cells bearing amino acid motif(PGLGGY) in their complementary-determining region 3 (CDR3) region were clonally expanding in ocular tissues on day 28 after immunization, although these T cells were not detected on day 14. These findings demonstrate that p518–529 reactive Th2-type T cells expand oligoclonally in the uveitic eyes in the late stage of EAU and may function as Th2-type suppressor T cells for improvement of the disease. [ABSTRACT FROM AUTHOR]

Cartilaginous fish express canonical B and T cell recognition genes, but their lymphoid organs and lymphocyte development have been poorly defined. Here, the expression of Ig, TCR, recombination-activating gene (Rag)-1 and terminal deoxynucleosidase (TdT) genes has been used to identify roles of various lymphoid tissues throughout development in the cartilaginous fish, Raja eglanteria (clearnose skate). In embryogenesis, Ig and TCR genes are sharply up-regulated at 8 weeks of development. At this stage TCR and TdT expression is limited to the thymus; later, TCR gene expression appears in peripheral sites in hatchlings and adults, suggesting that the thymus is a source of T cells as in mammals. B cell gene expression indicates more complex roles for the spleen and two special organs of cartilaginous fish—the Leydig and epigonal (gonad-associated) organs. In the adult, the Leydig organ is the site of the highest IgM and IgX expression. However, the spleen is the first site of IgM expression, while IgX is expressed first in gonad, liver, Leydig and even thymus. Distinctive spatiotemporal patterns of Ig light chain gene expression also are seen. A subset of Ig genes is pre-rearranged in the germline of the cartilaginous fish, making expression possible without rearrangement. To assess whether this allows differential developmental regulation, IgM and IgX heavy chain cDNA sequences from specific tissues and developmental stages have been compared with known germline-joined genomic sequences. Both non-productively rearranged genes and germline-joined genes are transcribed in the embryo and hatchling, but not in the adult. [ABSTRACT FROM PUBLISHER]

To understand the nature of long-term Th immune responses, we investigated in the present study the TCRBV gene repertoire of CD4+ T cells specific for the recall antigen tetanus toxoid (TT) in recipients of an allogeneic bone marrow transplantation (allo-BMT) at several time points after transplantation and in their BM donors. We observed that the TCR repertoire of TT-specific CD4+ Th cells was heterogeneous, and differed between allo-BMT recipients and their respective donors. Some individuals, however, used similar TCR–complementarity-determining region (CDR) 3 motifs that could reflect recognition of and selection by similar promiscuous epitopes of TT. Longitudinal analysis of this TT-specific T cell response revealed that T cells with completely identical TCR were present at several time points after the first analysis in allo-BMT recipients, most probably reflecting long-term stability of at least part of the antigen-specific TCR repertoire. Similar stability of the TT-specific TCR repertoire in time was also noted in the allo-BMT donors. These observations reveal that within a given individual the dominant antigen-specific T cell clones persist in time in an otherwise diverse TT-specific CD4+ T cell immune response. [ABSTRACT FROM PUBLISHER]

The T cell receptor (TCR) β chain transmembrane domain contains two evolutionarily conserved tyrosines (Y). In this study, the functional basis for the evolutionary conservation is addressed by mutation of the residues, expression of the mutants in hybridoma and primary T cells, and examination of TCR signaling function. We find that the phenotype of the mutants, both surface expression and ability to signal for IL-2 production, is highly variable in different mouse T hybridoma lines. Although we have not been able to determine the basis for these differences in the hybridomas, expression of the mutants in primary T cells provides a definitive assessment of mutant phenotype. We show that mutation of the N-terminal Y to either leucine (L) or alanine (A) results in low surface expression in primary T cells, while mutation of both N- and C-terminal Y to A or L abrogates surface expression. However, the more conservative mutation of both transmembrane Y to phenylalanine maintained receptor surface expression and assembly while severely disrupting signaling in primary T cells. Our data demonstrate that TCR β chain transmembrane Y are essential for TCR signal transduction as well as complex assembly. These findings suggest that protein–protein interactions involving membrane-spanning domains are likely relevant for TCR signal transduction mechanisms. [ABSTRACT FROM PUBLISHER]

VB17+ TCR dominate in Ni-driven T cell cultures from highly Ni-sensitized patients. Using transfection of TCR from three CD4+, VB17+, Ni-specific human T cell clones, we studied their Ni–MHC contacts by site-directed TCR mutation and combination of α and β chains between different TCR. All three TCR exhibited N-nucleotide-determined Arg–Asp motifs in their CDR3-β sequences. Two of them were specifically restricted to HLA-DR13, while the third one accepted a variety of HLA-DR alleles. The highly similar α or β chains of the DR13-restricted TCR were interchangable without loss of specificity, but α or β chains of other TCR were not tolerated. Mutations of their Arg–Asp motif revealed loss of reactivity upon exchanging Asp for Glu or Ala and of Arg for Ala but not of Arg for Lys or the Ni binding His. Reactivity was also destroyed by mutation of α chain position 51, proposed as a general contact site for MHC. Hence, in these two TCR the Arg–Asp motif is clearly involved in contacting Ni–MHC complexes, and close cooperation between α and β chain is required. In contrast, the third TCR retained Ni reactivity upon mutation of α chain position 51 or of its β chain Arg–Asp motif, which rather affected the pattern of DR cross-restriction. Moreover, its α chain paired with various β chains from other, even mouse TCR, irrespective of their specificity, retaining Ni reactivity as well as promiscuous HLA-DR restriction. This preponderance of an α chain in defining specificity indicates fundamental differences in Ni interactions of individual TCR and implies that β chain similarities may not necessarily result from antigen selection. [ABSTRACT FROM PUBLISHER]

Reactivity towards TCR peptides plays an important role in the regulation of several experimental autoimmune diseases. In a previous paper, we showed the TCRAV11 usage by an arthritogenic T cell clone isolated from a rat with adjuvant arthritis (AA). Moreover, we identified three immunogenic peptides in AV11: AV11 24–40, 41–55 and 66–80. In the present study, we show that T cells directed towards all three epitopes are part of the immune repertoire. The strongest delayed-type hypersensitivity (DTH) reaction was observed against the peptide derived from the third framework region, peptide AV11 66–80. DTH reactions to this peptide were detectable in naive rats and increased significantly after AA induction. Interestingly, modulation of the AV11 66–80 T cell response by nasal AV11 66–80 administration resulted in reduced DTH responses and in a strong inhibition of AA. These findings suggest that during the natural course of AA, T cells directed towards the third framework region of AV11 do not have a disease regulatory function, but instead play a role in the deterioration of AA. [ABSTRACT FROM PUBLISHER]

Myasthenia gravis (MG) is a T cell-regulated antibody-mediated autoimmune disease. Immunization with two myasthenogenic peptides, p195–212 and p259–271, that are sequences of the human acetylcholine receptor α subunit was shown to induce experimental autoimmune MG (EAMG)-associated immune responses. A peptide composed of the two altered peptide ligands (APL) of the myasthenogenic peptides (designated as dual APL) inhibited, in vitro and in vivo, those responses. The objectives of this study were to examine (i) whether in vivo T cell activation by p259–271 affects the cytokine profile and the T cell migration ability, and (ii) whether the latter are immunomodulated by in vivo administration of the dual APL. Our results showed that immunization of mice with p259–271 enriched the population of lymph node and spleen cells with subsets of T cells with strong adhesiveness towards E- and P-selectins. This enrichment was associated with an acquisition of a Th1-type cytokine profile. Treatment of the immunized mice with the dual APL interfered with both the migratory potential of the autoreactive T cells, and the production of the Th1-type cytokines IL-2 and IFN-γ (known to play a pathogenic role in MG and EAMG). T cells derived from APL-treated mice acquired a Th3-type cytokine profile, characterized by the secretion of the immunosuppresive cytokine transforming growth factor-β. Thus, our results suggest that T cell selectin ligands and T cell-derived cytokines are involved in the induction and immunomodulation of EAMG- and MG-associated T cell responses. [ABSTRACT FROM PUBLISHER]

Optimal T cell activation requires the interactions of co-stimulatory molecules, such as those in the CD28 and B7 protein families. Recently, we described the co-stimulatory properties of the murine ligand to ICOS, which we designated as B7RP-1. Here, we report the co-stimulation of human T cells through the human B7RP-1 and ICOS interaction. This ligand–receptor pair interacts with a KD ~ 33 nM and an off-rate with a t½ > 10 min. Interestingly, tumor necrosis factor (TNF)-α differentially regulates the expression of human B7RP-1 on B cells, monocytes and dendritic cells (DC). TNF-α enhances B7RP-1 expression on B cells and monocytes, while it inhibits it on DC. The human B7RP-1–Fc protein or cells that express membrane-bound B7RP-1 co-stimulate T cell proliferation in vitro. Specific cytokines, such as IFN-γ and IL-10, are induced by B7RP-1 co-stimulation. Although IL-2 levels are not significantly increased, B7RP-1 co-stimulation is dependent on IL-2. These experiments define the human ortholog to murine B7RP-1 and characterize its interaction with human ICOS. [ABSTRACT FROM PUBLISHER]

It is well known that IL-2 production of CD4+ T cells from old mice (old T cells) is impaired. In this study, we have examined TCR complex ζ chain expression of old T cells and their TCR downstream signal transduction pathways stimulated with anti-CD3. Activation of protein tyrosine kinases, Fyn and ZAP-70, and turnover of inositol phosphates stimulated with anti-CD3 were severely impaired in old T cells, although levels of these proteins were comparable to those in young T cells. Increase in intracellular Ca2+ concentration in old T cells was also impaired. Old T cells starting the Ca2+ oscillation by the anti-CD3 stimulation were severely decreased in number and the oscillation waves were broader in shape. T cells with ζ–FcɛRγ heterodimer in the TCR–CD3 complex were increased in proportion in old T cells with a concomitant decrease in the T cells with ζ-ζ homodimer. The density of the TCR–CD3 complex on old T cells was confirmed to be comparable to that on young T cells. The impairment in TCR downstream signal transduction pathways and the increase in ζ–FcɛRγ heterodimer in the TCR–CD3 complex were confirmed to be the situation in Th1 clones established from old mice. These results indicate that old T cells are impaired in response to TCR stimulation, because T cells with the TCR–CD3 complex containing the ζ–FcɛRγ heterodimer are increased in proportion in old T cells. [ABSTRACT FROM PUBLISHER]

Rheumatic heart disease (RHD) is a sequel of post-streptococcal throat infection. Molecular mimicry between streptococcal and heart components has been proposed as the triggering factor of the disease, and CD4+ T cells have been found predominantly at pathological sites in the heart of RHD patients. These infiltrating T cells are able to recognize streptococcal M protein peptides, involving mainly 1–25, 81–103 and 163–177 N-terminal amino acids residues. In the present work we focused on the TCR β chain family (TCR BV) usage and the degree of clonality assessed by β chain complementarity-determining region (CDR)-3 length analysis. We have shown that in chronic RHD patients, TCR BV usage in peripheral blood mononuclear cells (PBMC) paired with heart-infiltrating T cell lines (HIL) is not suggestive of a superantigen effect. Oligoclonal T cell expansions were more frequently observed in HIL than in PBMC. Some major BV expansions were shared between the mitral valve (Miv) and left atrium (LA) T cell lines, but an in-depth analysis of BJ segments usage in these shared expansions as well as nucleotide sequencing of the CDR3 regions suggested that different antigenic peptides could be predominantly recognized in the Miv and the myocardium. Since different antigenic proteins probably are constitutively represented in myocardium and valvular tissue, these findings could suggest a differential epitope recognition at the two lesional heart sites after a common initial bacterial challenge. [ABSTRACT FROM PUBLISHER]