Cell Receptor Gene Expression Research Articles

Binary outcomes of enhancer activity underlie stable random monoallelic expression.

Mitotically stable random monoallelic gene expression (RME) is documented for a small percentage of autosomal genes. We developed an in vivo genetic model to study the role of enhancers in RME using high-resolution single-cell analysis of natural killer (NK) cell receptor gene expression and enhancer deletions in the mouse germline. Enhancers of the RME NK receptor genes were accessible and enriched in H3K27ac on silent and active alleles alike in cells sorted according to allelic expression status, suggesting enhancer activation and gene expression status can be decoupled. In genes with multiple enhancers, enhancer deletion reduced gene expression frequency, in one instance converting the universally expressed gene encoding NKG2D into an RME gene, recapitulating all aspects of natural RME including mitotic stability of both the active and silent states. The results support the binary model of enhancer action, and suggest that RME is a consequence of general properties of gene regulation by enhancers rather than an RME-specific epigenetic program. Therefore, many and perhaps all genes may be subject to some degree of RME. Surprisingly, this was borne out by analysis of several genes that define different major hematopoietic lineages, that were previously thought to be universally expressed within those lineages: the genes encoding NKG2D, CD45, CD8α, and Thy-1. We propose that intrinsically probabilistic gene allele regulation is a general property of enhancer-controlled gene expression, with previously documented RME representing an extreme on a broad continuum.

Characterization and Monitoring of Antigen-Responsive T Cell Clones Using T Cell Receptor Gene Expression Analysis.

High-throughput T-cell receptor repertoire sequencing constitutes a powerful tool to study T cell responses at the clonal level. However, it does not give information on the functional phenotype of the responding clones and lacks a statistical framework for quantitative evaluation. To overcome this, we combined datasets from different experiments, all starting from the same blood samples. We used a novel, sensitive, UMI-based protocol to perform repertoire analysis on experimental replicates. Applying established bioinformatic routines for transcriptomic expression analysis we explored the dynamics of antigen-induced clonal expansion after in vitro stimulation, identified antigen-responsive clones, and confirmed their activation status using the expression of activation markers upon antigen re-challenge. We demonstrate that the addition of IL-4 after antigen stimulation drives the expansion of T cell clones encoding unique receptor sequences. We show that our approach represents a scalable, high-throughput immunological tool, which can be used to identify and characterize antigen-responsive T cells at clonal level.

Percutaneous BCG enhances innate effector antitumor cytotoxicity during treatment of bladder cancer: a translational clinical trial

ABSTRACT Background: Intravesical bacillus Calmette-Guérin (BCG) is the gold standard immunologic agent for treating patients with high-grade non-muscle invasive bladder cancer (NMIBC). Nevertheless, relapse rates remain high and BCG unresponsive NMIBC often requires bladder removal. Preclinical data suggest that priming with percutaneous BCG vaccine could improve response to intravesical BCG. Methods: A single-arm trial (NCT02326168) was performed to study the safety, immunogenicity, and preliminary efficacy of priming. Percutaneous BCG was given 21 days prior to intravesical BCG instillation in patients (n = 13) with high-risk NMIBC. Immune responses were monitored and compared to a sequentially enrolled cohort of nine control patients receiving only intravesical BCG. The effect of BCG on natural killer (NK) and γδ T cell in vitro cytotoxicity was tested. γδ T cell subsets were determined by T cell receptor gene expression with NanoString. Results: Priming was well tolerated and caused no grade ≥3 adverse events. The 3-month disease-free rate for prime patients was 85% (target goal ≥ 75%). Priming boosted BCG-specific immunity at 3 months and increased the activation status of in vitro expanded circulating NK and γδ T cells and their cytotoxicity against bladder cancer cells through receptor NKG2D. BCG enhanced the cytotoxicity of NK and γδ T cells against K562, T24, and UM-UC6 but not against UM-UC-3 or UM-UC-14 cells. Infiltrating γδ T cell subsets identified in the bladder includes γ9δ2 and γ8δ2. Conclusions: BCG priming is safe and tolerable. Poor sensitivity to NK and γδ T cell cytotoxicity by some bladder tumors represents a potential BCG-resistance mechanism.

Analysis of immunoglobulin and T cell receptor gene expression in ballan wrasse (Labrus bergylta) revealed an extraordinarily high IgM expression in the gut

The serum IgM concentration of ballan wrasse is relatively high, estimated to approximately 13 mg/ml in adult wild fish of 800 g. The present study revealed an unusual high abundance of IgM mRNA in the gut of ballan wrasse. Initially, transcripts encoding IgM, IgT, IgD, TCRα, TCRδ and CD3ε were quantified by RT-qPCR in several tissues of wild caught fish (approx. 800 g), indicating an elevated immune activity in hindgut and an extraordinarily high expression of IgM. Subsequently, a new RT-qPCR analysis was performed on the entire intestine, cut into four different segments, of reared fish (32–100 g). The analysis indicated immune activity along the entire intestine, but not as strong as in the hindgut. Furthermore, similar to the larger fish, the relative abundance of IgM transcripts was higher in the hindgut than in kidney and spleen, although the absolute level of IgM was in general higher in the larger fish. The secreted form of IgM was completely dominant in comparison to the membrane bound form of IgM and the other analysed genes. IgM was purified from gut mucus and external mucosal surfaces by magnetic beads coated with protein A. Mucus IgM reacted with rabbit antisera raised against serum IgM and contained subunits of the same size. Regarding the elevated immune activity in the intestine it is tempting to speculate on a possible compensatory strategy in this lineage of stomach-less fish, and that natural antibodies have an important role in the first line defence.

Lentiviral Vector Design for Optimal T Cell Receptor Gene Expression in the Transduction of Peripheral Blood Lymphocytes and Tumor-Infiltrating Lymphocytes

Lentiviral vectors containing promoters of distinct origins, that is, strong viral promoters (cytomegalovirus [CMV] and murine stem cell virus [MSCV]), a cellular promoter (phosphoglycerate kinase [PGK]), and two composite promoters (CAG [a composite promoter sequence comprised of the CMV enhancer and portions of the chicken beta-actin promoter and the rabbit beta-globin gene] and SV40/CD43), were used to evaluate green fluorescent protein (GFP) reporter gene expression in human primary peripheral blood lymphocytes (PBLs) and tumor-infiltrating lymphocytes (TILs). In PBLs, vectors containing the MSCV promoter were found to be optimal for expression in both minimally stimulated and highly activated lymphocytes. The stability of gene expression was monitored for up to 7 weeks in culture and the MSCV promoter-containing vector was found to be comparable to the cellular PGK promoter-containing vector. The MSCV promoter-containing lentiviral vector was also the most active in transduced TILs and these cells retained biological activity as measured by antimelanoma antigen reactivity. Using the knowledge gained in comparing individual promoters, a series of two-gene-containing lentiviral vectors was constructed in an attempt to produce the alpha and beta chains of antitumor antigen T cell receptors (TCRs). Dual-promoter or internal ribosome entry site (IRES)-containing vector designs were evaluated and found to be unable to produce both chains of the TCR in amounts that led to significant biological activity. In contrast, if the alpha and beta chains were linked by a 2A ribosomal skip peptide, both proper TCR chain pairing and biologically activity were observed. This paper emphasizes the need to optimize both promoter function and protein synthesis in constructs that require stoichiometric production of multiple protein subunits.

Gamma delta T cell receptor gene expression by muscle-infiltrating lymphocytes in the idiopathic inflammatory myopathies.

Autoreactive alpha beta T cells have been implicated as playing a primary pathogenic role in a group of diseases characterized by chronic muscle inflammation known as the idiopathic inflammatory myopathies (IIM). gamma delta T cells, a distinct and enigmatic class of T cells, play a less certain role in a variety of human autoimmune diseases including the IIM. In an attempt to understand the significance of gamma delta T cells in the IIM, we utilized a sensitive polymerase chain reaction (PCR) technique to evaluate gamma delta T cell receptor (TCR) gene expression in 45 muscle biopsies obtained from 42 IIM patients (17 polymyositis, 12 dermatomyositis, and 13 inclusion body myositis). gamma delta TCR gene expression was not detected in 36 specimens, the majority of muscle biopsies surveyed. gamma delta TCR gene expression by muscle-infiltrating lymphocytes was detected among nine clinically heterogeneous patients. We further analysed the junctional sequence composition of the V gamma 3 and V delta 1 transcripts, whose expression was prominent among gamma delta positive patients. DNA sequence analysis of V gamma 3 amplification products from two patients revealed the presence of several productively rearranged transcripts with amino acid sequence similarities within the V gamma 3-N-J gamma junctional domain. No amino acid sequence similarities were evident within the V delta-N-D delta-N-J delta region of V delta 1 transcripts amplified from four patients, although a distinct and dominant clonotype was detected from each patient. Our cumulative data suggest that unlike alpha beta T cells, gamma delta T cells do not play a prominent pathologic role in the IIM. In fact, the sporadic nature of gamma delta TCR gene expression detected among these patients implies that gamma delta T cell infiltration, when it occurs, is a secondary event perhaps resulting from non-specific inflammatory processes.

Differential TCR gene usage between WC1 − and WC1 + ruminant γδ T cell subpopulations including those responding to bacterial antigen

Ruminant gammadelta T cells are divided into subpopulations based on the presence or absence of WC1 co-receptors (scavenger-receptor-cysteine-rich family members uniquely expressed on gammadelta T cells). Evidence suggests WC1+ are inflammatory while WC1- are regulatory and that they also differ in their tissue distribution. Recently, this paradigm was refined further as cells that produce interferon-gamma and proliferate to autologous antigens, leptospira antigens, or IL-12 were largely found within the WC1+ subpopulation that bears the WC1.1 antigenic epitope but not that bearing the WC1.2 epitope. Here, the T cell receptor gene expression by these different subpopulations (WC1-, WC1.1+, and WC1.2+) was compared using flow cytometrically-purified cells and reverse transcriptase-polymerase chain reaction (RT-PCR). The WC1- gammadelta T cells had transcripts for all 11 possible combinations of the TRG subgroup V and C genes while those in both WC1+ subpopulations were restricted to TRGV3-TRGC5 and TRGV7-TRGC5. In contrast, all three subpopulations expressed transcripts from all four known bovine TRDV genes. Further analysis of the WC1+ gammadelta T cells that proliferated in leptospira antigen-stimulated cultures indicated that they do not represent a unique subpopulation within the larger WC1+ population based on their TCR gene usage. Moreover, sequencing of 65 transcripts showed that their junctional regions were diverse as TRGJ5-1, TRGJ5-2, TRDJ1, and TRDJ3 were used, and CDR3s ranged from 9 to 24 amino acids. The restricted but shared gammadelta TCR gene usage for WC1.1+, WC1.2+, and WC1(+)-antigen-responsive cells leaves open the possibility that the WC1 co-receptor is an important determining element in the activation process and subsequent response.

Gene expression profiles in microdissected neurons from human hippocampal subregions

Pyramidal neurons in hippocampal subregions are selectively vulnerable in certain disease states. To investigate, we tested the hypothesis that selective vulnerability in human hippocampus is related to regional differences in neuronal cell death and cell receptor gene expression in CA1 vs. CA3 subregions. We used laser capture microdissection to remove approximately 600 CA1 and 600 CA3 pyramidal neurons each from five fresh-frozen normal post-mortem brains, extracted total RNA and double-amplified mRNA. This was reverse transcribed and labeled for hybridization onto human cDNA array chips containing probes to 10,174 genes and unknown ESTs. RNA from additional microdissections was pooled for replicate hybridizations and quantitative RT-PCR validation. Gene expression differences were few (<1%). We found 43 enriched genes in CA1 neuronal samples that included peripheral benzodiazipine receptor-associated protein, nicotinic cholinergic receptor, two chemokine receptors (CCR1 and CCR5) and several transcriptional factors. We found 17 enriched genes in the CA3 neuronal samples that included fibroblast growth factor receptor and prostaglandin-endoperoxide synthase 1. We found no differential gene expression for 23 calcium channel proteins; nine transporter proteins; 55 cell death and apoptotic regulator proteins; and an additional 497 cell receptors, including 24 glutamate receptors. Quantitative RT-PCR of four differentially expressed genes confirmed the microarray data. The results confirm the ability to examine gene expression profiles in microdissected neurons from human autopsy brain. They show only minor gene expression differences between two distinct neuronal populations in the hippocampus and suggest that selective hippocampal vulnerability is due to factors other than intrinsic differential expression in glutamate receptors and cell death genes.

No restriction of T cell receptor gene expression by lamina propria lymphocytes in Crohnʼs disease

No restriction of T cell receptor gene expression by lamina propria lymphocytes in Crohnʼs disease

Evaluating human T cell receptor gene expression by PCR.

This unit describes the use of PCR to characterize and quantify rearranged transcripts from specific T cell receptor variable gene families in human tissue and peripheral blood lymphocytes. The strategy outlined in this unit has been extensively used on different sources of human tissue including brain, spinal cord, and skeletal muscle. A protocol is provided to clone and sequence PCR-amplified cDNA transcripts to study the junctional diversity of the expressed genes. A support protocol describes a method for reverse transcribing total RNA to make the cDNA required by the other protocols.

T cell-mediated immune mechanisms in myositis.

Advances in molecular biologic techniques and the availability of novel immunologic reagents have allowed new approaches to understanding the pathogenesis of human autoimmune diseases, including the idiopathic inflammatory myopathies. Indirect evidence that autoreactive T cells mediate muscle inflammation in the human myositis syndromes has been strengthened by recent studies describing restricted T cell receptor gene expression in certain clinical and/or serologic groups of myositis patients. These findings are supported by other investigations documenting abnormal patterns of cytokine, adhesion molecule, and major histocompatibility complex antigen expression within inflammatory lesions. The major challenge of future studies is to identify the specific antigen(s) responsible for initiating and perpetuating these harmful immune responses.

Induction of autoimmune disease in mice by germline alteration of the T cell receptor gene expression.

Germline expression of rearranged TCR alpha-chain transgenes with the Ig H chain enhancer reproducibly elicits T cell-mediated autoimmune disease in the thyroid gland, gastric mucosa, Langerhans islets, salivary gland, ovaries, and testes in selected strains of normal mice. Multiple organs are destroyed in a single transgenic mouse and the same organ in transgenic strains with different MHC background, suggesting the transgene expression can elicit self-reactive T cell clones having different Ag specificities and MHC restrictions. Construction of this autoimmune-inducing TCR alpha EH transgene does not require particular V alpha J alpha gene segments or Ag specificities. Moreover, the autoimmune disease can be adoptively transferred to syngeneic normal mice by T cells expressing endogenous TCR alpha-chains. Taken together, these results indicate that the TCR alpha EH transgene expression does not suppress endogenous alpha-chain gene rearrangement and may trigger the expansion/activation of various self-reactive T cells expressing endogenous TCR alpha- and beta-chains. Furthermore, it appears that the transgene-induced autoimmune T cells are not deleted in the normal thymus or rendered anergic upon contact with the normal target self Ag, but can be controlled by a T cell-dependent mechanism, since transfer of the transgenic bone marrow cells to histocompatible SCID mice produces the same autoimmune disease as in the donors, and the autoimmune development in the SCID mice is effectively prevented by co-transfer of syngeneic nontransgenic T cells. This novel autoimmune model produced by genetic manipulation of the T cell lineage, not the target self Ag or the environment of T cell differentiation/selection, should be useful for elucidating the immunologic and genetic basis of autoimmune disease.

CYTOKINE AND T CELL RECEPTOR GENE EXPRESSION AT THE SITE OF ALLOGRAFT REJECTION1

Intragraft cytokine and T cell receptor gene expression was analyzed in rejecting renal allografts by polymerase chain reaction (PCR). Message for IL-1 beta, IL-6, and TNF-alpha was detected in nephrectomy tissue with pathological evidence of acute or chronic rejection. Similarly, mRNA for both IL-6 and TNF-alpha was present in renal biopsies from acute rejecting kidneys. IL-2R, IL-4, and IL-5 mRNA was present in both rejecting and rejected kidney allografts, indicating that these cytokines may play a role in ongoing renal allograft rejection. Conversely, IL-2, IL-7, and IFN-gamma message was detected infrequently. In order to address the diversity of T cells in rejecting kidneys, we have analyzed the clonality of the TcR present within the allograft tissue. Rearranged TcR genes were identified in all allografts examined (n = 16) indicating the presence of T cells bearing the alpha/beta TcR. We have determined that there is a heterogeneous infiltration of T cells in the rejected allograft with TcR representing x = 7.47 +/- 2.4 families rearranged in samples obtained from nephrectomies, whereas x = 5.33 +/- 0.58 families were detected in samples obtained from biopsy tissue. These data indicate that (1) cytokines are produced locally which may contribute to graft cell destruction, (2) the heterogeneity of intragraft T cells during kidney allograft rejection may exist because nonspecific lymphocytes have been recruited to the site by locally produced cytokines or because T cells are responding to multiple epitopes or multiple donor antigens. Detection of intragraft cytokines and TcR may prove useful in elucidating the mechanism of rejection and therefore lead to improved immunosuppression.

T cell receptor gene expression in sheep: differential usage of TcR1 in the periphery and thymus.

We have isolated cDNA clones for each chain of the sheep T cell receptor (TcR) and used these to analyze TcR gene expression in thymocyte and peripheral T cell subsets. Outer cortical thymocytes expressed a low level of mature message for all TcR chains suggesting that intrathymic precursors for the alpha/beta and gamma/delta lineages occur in this population. Inner cortical and medullary thymocytes expressed high levels of mature alpha/beta transcripts, low levels of mature delta transcripts but no detectable gamma message. Mature alpha/beta transcripts were detected in peripheral CD4+ lymphocytes and these as well as CD8+ cells expressed a surface heterodimer of 85 kDa which resolved into 40- and 50-kDa subunits after reduction. Peripheral CD4-CD8-lymphocytes, which in sheep are marked by the T19 antigen and may account for up to 60% of T cells in blood, expressed a surface heterodimer of 75 kDa. The T19+ cells had high levels of mature delta and truncated beta transcripts in their cytoplasm but did not express the C gamma gene detected in DN thymocytes, although they seem to share V gamma and/or J gamma elements. Both forms of the sheep TcR are associated with CD3 molecules on the cell surface. These results show that (a) in contrast to the situation in rodents and humans, a large proportion of peripheral sheep lymphocytes use TcR gamma/delta; (b) the proportion of T cells in the periphery which use TcR gamma/delta is greater than in thymus; and (c) CD4-CD8- cells in thymus and periphery (T19+) use the same C delta gene but appear to use different C gamma genes, suggesting that in sheep there may be more than one lineage of lymphocytes expressing TcR gamma/delta.

Thymocyte development: an analysis of T cell receptor gene expression in 519 newborn thymocyte hybridomas.

We have examined the frequency of expression of individual alpha and beta chain V gene families in a population of immature T cells that has not been selected or tolerized. To accomplish this, we generated 519 T cell hybridomas from freshly isolated thymocytes of newborn C57BL/10 mice and subjected RNA from these hybrids to hybridization analysis with 11 V alpha, 16 V beta, C gamma and C delta probes. Comparison of the expressed repertoire of V beta gene segments in this newborn thymocyte population with similar data previously generated from adult peripheral T cells (Bill et al., Proc. Natl. Acad. Sci. USA 1988. 85: 9184.) revealed two V beta genes, V beta 12 and V beta 15, whose expression is decreased in the periphery possibly due to the effects of tolerance. An additional V beta gene segment (V beta 10) and a V beta gene family (V beta 5.1, V beta 5.2) were expressed more frequently in the mature, peripheral population than in the newborn thymus. These findings may represent two instances of positive selection of T cells. Furthermore, unlike VH gene segments, D beta-proximal V beta genes are not overrepresented on this collection of immature thymocyte hybridomas. A similar analysis of V alpha gene family expression was hampered by the fact that V alpha gene segments were expressed in only 15% of newborn thymocyte hybridomas (compared to 58% of adult hybridomas). An unexpectedly large fraction (57%) of those newborn hybrids expressing a V alpha gene segment was also found to express C delta mRNA and further examination revealed that several V alpha gene probes were actually detecting delta chain mRNA. The most notable of these was the V alpha 7 gene family, which accounted for approximately one-third of the expressed V alpha genes but was expressed exclusively as part of a delta chain mRNA. We found no examples of hybridomas co-expressing both full-length alpha and delta chain mRNA, despite significant numbers of hybridomas co-expressing full-length beta and gamma chain transcripts. This observation suggests that a large number of mature alpha/beta T cells may have proceeded through development without having gone through a stage where a delta chain message is expressed, thereby precluding their development into gamma/delta cells.

Development of T3/T cell receptor gene expression in human pre-T neoplasms

Acquisition of mature T cell function and the T cell antigen receptor repertoire occur in the thymus. In an effort to delineate the cascade of events leading to T cell maturation, we analyzed a series of clonal human precursor T cell neoplasms representing early, middle, and late stages of intrathymic differentiation. Rearrangements of the T cell receptor beta and gamma genes appear concurrently and are preceded by surface expression of the 3A1 (CD7) molecule. Subsequent transcription of the beta gene is coordinated with surface expression of T1 (CD5) and T11 (CD2), transcription of T3 delta mRNA, and the appearance of intracellular T3 (CD3) protein. As late events, T alpha gene transcripts appear and, finally, T3, the multichain complex linked to the T cell receptor, is presented on the cell surface. Findings reported here provide a model of the developmental orchestration of genes encoding antigen recognition in human T cells.

Development of T3/T Cell Receptor Gene Expression in Human Pre-T Neoplasms

Acquisition of mature T cell function and the T cell antigen receptor repertoire occur in the thymus. In an effort to delineate the cascade of events leading to T cell maturation, we analyzed a series of clonal human precursor T cell neoplasms representing early, middle, and late stages of intrathymic differentiation. Rearrangements of the T cell receptor beta and gamma genes appear concurrently and are preceded by surface expression of the 3A1 (CD7) molecule. Subsequent transcription of the beta gene is coordinated with surface expression of T1 (CD5) and T11 (CD2), transcription of T3 delta mRNA, and the appearance of intracellular T3 (CD3) protein. As late events, T alpha gene transcripts appear and, finally, T3, the multichain complex linked to the T cell receptor, is presented on the cell surface. Findings reported here provide a model of the developmental orchestration of genes encoding antigen recognition in human T cells.

T cell receptor gene expression in autoimmune mice.

Autoimmunity in mice with the lpr/lpr and gld/gld genotypes is accompanied by profound lymphadenopathy characterized by the presence of a massive expansion of an unusual T cell subset. The abnormal lymph node T cells were found to express TcR beta and TcR alpha transcripts of expected sizes. There was a 10-fold increase in the 1.3-kb TcR beta transcript and a twofold increase in TcR alpha gene expression, even though Thy-1 expression was in general similar to controls. A study of T cell receptor expression during ontogeny failed to reveal any striking differences between lpr/lpr and congenic mice. There was a strong correlation between TcR beta expression and c-myb expression; however, there was no necessary association of TcR beta and c-myb expression when various T cell lines were examined. Background genes were found to influence the expression of T cell receptor genes in lpr/lpr mice. AKR-lpr/lpr lymph node cells, but not cells from other lpr/lpr mice or AKR +/+ mice, had the predominance of the 1.0-kb TcR beta transcript, which represents the nonfunctional D-J TcR beta rearrangements. Lymph nodes from MRL-lpr/lpr mice, but not C57BL/6-lpr/lpr or AKR-lpr/lpr mice, were found to express small amounts of the TcR gamma transcript. In addition, MRL-lpr/lpr but not C57BL/6-lpr/lpr mice had an age-related decrease in thymic TcR beta expression along with a decrease in thymic c-myb expression. The current study extends the characterization of T cell gene expression abnormalities in peripheral T cells of gld/gld and lpr/lpr and describes certain similarities of these cells to immature thymocytes at a molecular level. Furthermore, it illustrates the complex interactions between "background genes" and genes responsible for lymphoproliferation, which in concert lead to specific molecular and cellular abnormalities.