High-throughput single-cell sequencing of paired TCRα and TCRß genes for the direct expression-cloning and functional analysis of murine T-cell receptors.

Precise clonal and functional assessments of the T cell receptor (TCR) repertoire diversity require paired TCRα and TCRß gene sequence information at monoclonal level. However, available single-cell strategies are typically limited in throughput and often do not provide full-length DNA templates for direct gene cloning. Here, we describe a high-throughput strategy for the unbiased amplification and automated sequence analysis of paired TCRα and TCRß genes from primary mouse T cells. The platform links cell phenotype and TCR gene sequence information at single-cell level. Furthermore, it enables direct functional analyses through the efficient cloning of both genes and the generation of stable TCR expressing cell lines. This highly efficient workflow is a powerful tool to determine the diversity and quality of the murine T-cell repertoire in various settings, for example in vaccine development, infectious diseases, autoimmunity, or cancer.

Targeting CD123 in acute myeloid leukemia using a T-cell-directed dual-affinity retargeting platform.

T-cell-directed killing of tumor cells using bispecific antibodies is a promising approach for the treatment of hematologic malignancies. Here we describe our preclinical work with a dual-affinity retargeting (DART) molecule generated from antibodies to CD3 and CD123, designed to redirect T cells against acute myeloid leukemia blasts. The CD3×CD123 DART (also referred to as MGD006/S80880) consists of 2 independent polypeptides, each composed of the VH of 1 antibody in tandem with the VL of the other antibody. The target antigen CD123 (interleukin 3RA) is highly and differentially expressed in acute myeloid leukemia (AML) blasts compared with normal hematopoietic stem and progenitor cells. In this study we demonstrate that the CD3×CD123 DART binds to both human CD3 and CD123 to mediate target-effector cell association, T-cell activation, proliferation, and receptor diversification. The CD3×CD123 DART also induces a dose-dependent killing of AML cell lines and primary AML blasts in vitro and in vivo. These results provide the basis for testing the CD3×CD123 DART in the treatment of patients with CD123(+) AML.

Formation of dynamic gamma-H2AX domains along broken DNA strands is distinctly regulated by ATM and MDC1 and dependent upon H2AX densities in chromatin.

A hallmark of the cellular response to DNA double-strand breaks (DSBs) is histone H2AX phosphorylation in chromatin to generate gamma-H2AX. Here, we demonstrate that gamma-H2AX densities increase transiently along DNA strands as they are broken and repaired in G1 phase cells. The region across which gamma-H2AX forms does not spread as DSBs persist; rather, gamma-H2AX densities equilibrate at distinct levels within a fixed distance from DNA ends. Although both ATM and DNA-PKcs generate gamma-H2AX, only ATM promotes gamma-H2AX formation to maximal distance and maintains gamma-H2AX densities. MDC1 is essential for gamma-H2AX formation at high densities near DSBs, but not for generation of gamma-H2AX over distal sequences. Reduced H2AX levels in chromatin impair the density, but not the distance, of gamma-H2AX formed. Our data suggest that H2AX fuels a gamma-H2AX self-reinforcing mechanism that retains MDC1 and activated ATM in chromatin near DSBs and promotes continued local phosphorylation of H2AX.

Loss of T cells influences sex differences in behavior and brain structure.

Clinical and animal studies demonstrate that immune-brain communication influences behavior and brain function. Mice lacking T cell receptor ß and δ chains were tested in the elevated plus maze, open field, and light-dark test and showed reduced anxiety-like behavior compared to wild type. Interestingly sex differences were observed in the behavioural phenotype of TCRß-/-δ- mice. Specifically, female TCRß-/-δ- mice spent more time in the light chamber compared to wild type females, whereas male TCRß-/-δ- spent more time in the center of the open field compared to wild type males. In addition, TCRß-/-δ- mice did not show sex differences in activity-related behaviors observed in WT mice. Ex vivo brain imaging (7 Tesla MRI) revealed volume changes in hippocampus, hypothalamus, amygdala, periaqueductal gray, and dorsal raphe and other brain regions between wild type and T cell receptor knockout mice. There was also a loss of sexual dimorphism in brain volume in the bed nucleus of the stria terminalis, normally the most sexually dimorphic region in the brain, in immune compromised mice. These data demonstrate the presence of T cells is important in the development of sex differences in CNS circuitry and behavior.

53BP1 facilitates long-range DNA end-joining during V(D)J recombination.

Variable, diversity and joining (V(D)J) recombination and class-switch recombination use overlapping but distinct non-homologous end joining pathways to repair DNA double-strand-break intermediates. 53BP1 is a DNA-damage-response protein that is rapidly recruited to sites of chromosomal double-strand breaks, where it seems to function in a subset of ataxia telangiectasia mutated (ATM) kinase-, H2A histone family member X (H2AX, also known as H2AFX)- and mediator of DNA damage checkpoint 1 (MDC1)-dependent events. A 53BP1-dependent end-joining pathway has been described that is dispensable for V(D)J recombination but essential for class-switch recombination. Here we report a previously unrecognized defect in the joining phase of V(D)J recombination in 53BP1-deficient lymphocytes that is distinct from that found in classical non-homologous-end-joining-, H2ax-, Mdc1- and Atm-deficient mice. Absence of 53BP1 leads to impairment of distal V-DJ joining with extensive degradation of unrepaired coding ends and episomal signal joint reintegration at V(D)J junctions. This results in apoptosis, loss of T-cell receptor alpha locus integrity and lymphopenia. Further impairment of the apoptotic checkpoint causes propagation of lymphocytes that have antigen receptor breaks. These data suggest a more general role for 53BP1 in maintaining genomic stability during long-range joining of DNA breaks.

Role of TRAV locus in low caries experience.

Caries is the most common chronic, multifactorial disease in the world today; and little is still known about the genetic factors influencing susceptibility. Our previous genome-wide linkage scan has identified five loci related to caries susceptibility: 5q13.3, 13q31.1, 14q11.2, 14q 24.3, and Xq27. In the present study, we fine mapped the 14q11.2 locus to identify genetic contributors to caries susceptibility. Four hundred seventy-seven subjects from 72 pedigrees with similar cultural and behavioral habits and limited access to dental care living in the Philippines were studied. An additional 387 DNA samples from unrelated individuals were used to determine allele frequencies. For replication purposes, a total of 1,446 independent subjects from four different populations were analyzed based on their caries experience (low versus high). Forty-eight markers in 14q11.2 were genotyped using TaqMan chemistry. Transmission disequilibrium test was used to detect over transmission of alleles in the Filipino families, and Chi-square, Fisher's exact and logistic regression were used to test for association between low caries experience and variant alleles in the replication data sets. We finally assessed the mRNA expression of TRAV4 in the saliva of 143 study subjects. In the Filipino families, statistically significant associations were found between low caries experience and markers in TRAV4. We were able to replicate these results in the populations studied that were characteristically from underserved areas. Direct sequencing of 22 subjects carrying the associated alleles detects one missense mutation (Y30R) that is predicted to be probably damaging. Finally, we observed higher expression in children and teenagers with low caries experience, correlating with specific alleles in TRAV4. Our results suggest that TRAV4 may have a role in protecting against caries.

Long-distance regulation of fetal V(δ) gene segment TRDV4 by the Tcrd enhancer.

Murine Tcra and Tcrd gene segments are organized into a single genetic locus (Tcra/Tcrd locus) that undergoes V(D)J recombination in CD4(-)CD8(-) double-negative (DN) thymocytes to assemble Tcrd genes and in CD4(+)CD8(+) double-positive thymocytes to assemble Tcra genes. Recombination events are regulated by two developmental stage-specific enhancers, E(δ) and E(α). Effects of E(α) on Trca/Tcrd locus chromatin have been well documented, but effects of E(δ) have not. In this regard, E(α) acts over long distances to activate many V(α) and J(α) segments for recombination in double-positive thymocytes. However, in DN thymocytes, it is unclear whether E(δ) functions over long distances to regulate V(δ) gene segments or functions only locally to regulate D(δ) and J(δ) gene segments. In this study, we analyzed germline transcription, histone modifications, and recombination on wild-type and E(δ)-deficient alleles in adult and fetal thymocytes. We found that E(δ) functions as a local enhancer whose influence is limited to no more than ∼10 kb in either direction (including D(δ), J(δ), and TRDV5 gene segments) in adult DN thymocytes. However, we identified a unique long-distance role for E(δ) promoting accessibility and recombination of fetal V(δ) gene segment TRDV4, over a distance of 55 kb, in fetal thymocytes. TRDV4 recombination is specifically repressed in adult thymocytes. We found that this repression is enforced by a developmentally regulated loss of histone acetylation. Constitutively high levels of a suppressive modification, histone H3 lysine 9 dimethylation, may contribute to repression as well.

A structural basis for selection and cross-species reactivity of the semi-invariant NKT cell receptor in CD1d/glycolipid recognition.

Little is known regarding the basis for selection of the semi-invariant alphabeta T cell receptor (TCR) expressed by natural killer T (NKT) cells or how this mediates recognition of CD1d-glycolipid complexes. We have determined the structures of two human NKT TCRs that differ in their CDR3beta composition and length. Both TCRs contain a conserved, positively charged pocket at the ligand interface that is lined by residues from the invariant TCR alpha- and semi-invariant beta-chains. The cavity is centrally located and ideally suited to interact with the exposed glycosyl head group of glycolipid antigens. Sequences common to mouse and human invariant NKT TCRs reveal a contiguous conserved "hot spot" that provides a basis for the reactivity of NKT cells across species. Structural and functional data suggest that the CDR3beta loop provides a plasticity mechanism that accommodates recognition of a variety of glycolipid antigens presented by CD1d. We propose a model of NKT TCR-CD1d-glycolipid interaction in which the invariant CDR3alpha loop is predicted to play a major role in determining the inherent bias toward CD1d. The findings define a structural basis for the selection of the semi-invariant alphabeta TCR and the unique antigen specificity of NKT cells.

Selected murine residues endow human TCR with enhanced tumor recognition.

TCR-gene transfer can mediate tumor regression in terminally ill melanoma patients. However, the formation of mix dimers between endogenous and transduced TCR chains may result in the surface dilution of the introduced TCR, which translates in poorer cellular avidity. Recently, we reported that murinization of human TCRs (i.e., the replacement of human C regions by murine ones) can improve TCR function. However, because xenogenic sequences may trigger immunogenicity, we sought to identify the essential murine residues that mediate this enhanced functional effect. We constructed murine/human chimeras of alpha- and beta-chains and assessed for their surface expression and function. We identified an evolutionary-unique lysine residue in Cbeta, central to murine TCR function. The mapping of Calpha revealed that a few short stretches of amino acids play a role in enhancing TCR function, one of the most important ones being the SDVP sequence. This information led us to design improved and minimally murinized human TCR C regions that mediate increased tumor recognition. This also enabled us to suggest a structural model that could explain the role of the aforementioned residues in promoting the preferential pairing and stability of murinized TCRs. Overall, these findings could have implications for the treatment of malignant diseases using TCR-gene transfer.

Differential utilization of T cell receptor TCR alpha/TCR delta locus variable region gene segments is mediated by accessibility.

T cell receptor (TCR) variable region exons are assembled from germline V, (D), and J gene segments, each of which is flanked by recombination signal (RS) sequences that are composed of a conserved heptamer, a spacer of 12 or 23 bp, and a characteristic nonamer. V(D)J recombination only occurs between V, D, and J segments flanked by RS sequences that contain, respectively, 12(12-RS)- and 23(23-RS)-bp spacers (12/23 rule). Additional mechanisms can restrict joining of 12/23 RS matched segments beyond the 12/23 rule (B12/23). The TCRdelta locus is contained within the TCRalpha locus; TCRalpha variable region exons are encoded by TRAV and TRAJ segments and those of TCRdelta by TRDV, TRDD, and TRDJ segments. On the basis of the 12/23 rule, both TRAV and TRDV gene segments are compatible to rearrange with TRDD gene segments; however, TRAV-to-TRDD joins are not observed in vivo. Absence of TRAV-to-TRDD rearrangement might be explained either by B12/23 restriction or by differential accessibility of the TRDV versus TRAV gene segments for rearrangement to TRDD. We used in vitro substrate analysis to reveal that both TRAV and TRDV 23-RSs mediate rearrangements to the 5'TRDD1 12-RS, demonstrating that B12/23 restriction does not explain these rearrangement biases. However, targeted replacement of TRDD1 and its 12-RSs with TRAJ38 and its 12-RS showed that TRDV gene segments rearrange with the ectopic TRAJ38, whereas TRAV segments do not. Our results demonstrate that sorting of TRAV and TRDV gene segments is determined by differential locus accessibility during T cell development.

Regulation of TCR delta and alpha repertoires by local and long-distance control of variable gene segment chromatin structure.

Murine Tcrd and Tcra gene segments reside in a single genetic locus and undergo recombination in CD4- CD8- (double negative [DN]) and CD4+ CD8+ (double positive [DP]) thymocytes, respectively. TcraTcrd locus variable gene segments are subject to complex regulation. Only a small subset of approximately 100 variable gene segments contributes substantially to the adult TCRdelta repertoire. Moreover, although most contribute to the TCRalpha repertoire, variable gene segments that are Jalpha proximal are preferentially used during primary Tcra recombination. We investigate the role of local chromatin accessibility in determining the developmental pattern of TcraTcrd locus variable gene segment recombination. We find variable gene segments to be heterogeneous with respect to acetylation of histones H3 and H4. Those that dominate the adult TCRdelta repertoire are hyperacetylated in DN thymocytes, independent of their position in the locus. Moreover, proximal variable gene segments show dramatic increases in histone acetylation and germline transcription in DP thymocytes, a result of super long-distance regulation by the Tcra enhancer. Our results imply that differences in chromatin accessibility contribute to biases in TcraTcrd locus variable gene segment recombination in DN and DP thymocytes and extend the distance over which the Tcra enhancer can regulate chromatin structure to a remarkable 525 kb.

Flexible stereospecific interactions and composition within nucleoprotein complexes assembled on the TCR alpha gene enhancer.

During thymocyte maturation, enhancers of genes encoding for TCRdelta (Tcrd) and TCRalpha (Tcra), Edelta(8), and Ealpha, work as a developmental switch controlling transition from Tcrd to Tcra activity at the Tcrad locus. Previous experiments revealed that an Ealpha fragment, Talpha1-Talpha2, which constitutes a well-characterized compact nucleoprotein structure led to premature activation of V(D)J recombination compared with that observed for the entire Ealpha or Talpha1-Talpha4. These experiments indicated that Talpha3-Talpha4 collaborates with factors bound to Talpha1-Talpha2 for the strict developmental regulation of Tcra rearrangement. The compact enhanceosome created on Talpha1-Talpha2 explained the molecular basis for requirement of intact Talpha2 TCF/LEF and ets sites for enhancer function. We have created a mutant version of Ealpha, EalphaMC, in which Edelta myb and runx sites have been substituted for Talpha2 runx and ets sites, that argues against the notion of a requirement for strict Ealpha enhanceosome structure for function. EalphaMC resulted in a very potent enhancer indicating that stereospecific interactions among proteins that form an Ealpha enhanceosome are rather flexible. Activation of V(D)J recombination by EalphaMC during thymocyte development resulted, however, to be premature and indistinguishable from that of Talpha1-Talpha2. These results indicate that Talpha3-Talpha4 itself is not sufficient to impart a developmental delay to a chimeric "early" enhancer, and indicate the need for functional collaboration between Talpha2 runx/ets sites binding proteins and proteins bound to Talpha3-Talpha4 for proper developmental activation. The possibility of assembly of distinct sets of proteins on Ealpha might represent a more flexible form of information processing during thymocyte development.

Correlation between TCRCalpha -560 C/T polymorphism and the clinical presentation of Uygur IgA nephropathy patients in XinJiang.

OBJECTIVE: To investigate the relationship between T cell receptor alpha chain constant gene (TCRCalpha) -560 C/T polymorphism and the clinical presentation of Uygur IgA Nephropathy patients in XinJiang. METHODS: TCRCalpha -560 C/T genotypes were determined by PCR-RFLP in 300 Chinese Uygur IgAN patients and 600 healthy Chinese Uygur control subjects. All subjects were classified, based on their genotype, into TT, CT and CC groups and their corresponding clinical presentation was analyzed. RESULTS: No significant difference was observed in the frequency of CC/CT/TT genotypes in patients and control subjects (chi2 = 0.904, P = 0.636). However, the incidence of intermittent microscopic hematuria and proteinuria is significantly higher in patients with CT genotype than CC and TT genotypes (chi2 = 33.978, P < 0.05). CONCLUSION: TCRCalpha-560 C/T gene polymerphism may be associated with the occurrence of intermittent microscopic hematuria and proteinuria in Chinese Uygur IgAN patients.

Altered production of immunoregulatory cytokines by invariant Valpha19 TCR-bearing cells dependent on the duration and intensity of TCR engagement.

Cells bearing invariant Valpha19-Jalpha33 TCR alpha chains are believed to participate in the regulation of inflammatory autoimmune diseases. In this study, the potential to produce immunoregulatory cytokines by these cells was characterized in order to find the mechanism underlying their immunoregulatory functions. Serum levels of IL-4, IL-10, transforming growth factor-beta, IFN-gamma and IL-17 increased in mice over-expressing an invariant Valpha19-Jalpha33 TCR alpha transgene (Valpha19 Tg) in response to anti-CD3 antibody injection. NK1.1(+) Valpha19 Tg(+), but not NK1.1(-) Valpha19 Tg(+) cells, promptly produced immunoregulatory IL-4, IFN-gamma and IL-17 upon invariant TCR engagement with immobilized anti-CD3 antibody in culture. The activation of Valpha19 Tg(+) cells then triggered the production of pro-inflammatory cytokines by bystander cells. Interestingly, the ratio of T(h)2 cytokines such as IL-4, IL-5 and IL-10, but not pro-inflammatory IL-17, to IFN-gamma was increased when the intensity of the stimulation to invariant TCR was attenuated. Collectively, these findings suggest that invariant Valpha19 TCR(+) cells have the potential to participate in the regulation of inflammatory autoimmunity by producing T(h)2-biased cytokines in certain circumstances.

IMGT® Biocuration and Comparative Analysis of Bos taurus and Ovis aries TRA/TRD Loci.

The adaptive immune response provides the vertebrate immune system with the ability to recognize and remember specific pathogens to generate immunity, and mount stronger attacks each time the pathogen is encountered. T cell receptors are the antigen receptors of the adaptive immune response expressed by T cells, which specifically recognize processed antigens, presented as peptides by the highly polymorphic major histocompatibility (MH) proteins. T cell receptors (TR) are divided into two groups, αß and γδ, which express distinct TR containing either α and ß, or γ and δ chains, respectively. The TRα locus (TRA) and TRδ locus (TRD) of bovine (Bos taurus) and the sheep (Ovis aries) have recently been described and annotated by IMGT® biocurators. The aim of the present study is to present the results of the biocuration and to compare the genes of the TRA/TRD loci among these ruminant species based on the Homo sapiens repertoire. The comparative analysis shows similarities but also differences, including the fact that these two species have a TRA/TRD locus about three times larger than that of humans and therefore have many more genes which may demonstrate duplications and/or deletions during evolution.

Rapid and reliable generation of invariant natural killer T-cell lines in vitro.

Several tools have proved useful in the study of invariant natural killer T (iNKT) cells, including CD1d-deficient mice, J alpha281-deficient mice, synthetic lipid antigens and antigen-loaded CD1d tetramers. However, the generation and examination of long-term primary murine iNKT cell lines in vitro has been challenging. Here, we show the rapid generation of iNKT cell lines from splenic iNKT cells of V alpha14 T-cell receptor (TCR) transgenic (Tg) mice. These purified iNKT cells were stimulated by bone marrow-derived dendritic cells (BMDCs) loaded with alpha-galactosylceramide (alphaGalCer) and cultured with interleukin (IL)-2 and IL-7. iNKT cells proliferated dramatically, and the cell number exhibited a 100-fold increase within 2 weeks and a 10(5)-fold increase in 8 weeks after repeated stimulation with alphaGalCer. The iNKT cell lines consisted of iNKT cells expressing V beta chains including V beta8.1/8.2, V beta14, V beta10, V beta6 and V beta7, and responded to stimulation with alphaGalCer presented both by BMDCs and by plate-bound CD1d. In addition, the iNKT cell lines produced interferon (IFN)-gamma when activated by lipopolysaccharide (LPS) or CpG oligodeoxynucleotide (ODN)-stimulated BMDCs. Further, we show that iNKT cell lines produced cytokines in response to microbial antigens. In summary, high-yield iNKT cell lines were generated very rapidly and robustly expanded, and these iNKT cells responded to both TCR and cytokine stimulation in vitro. Given the desire to study primary iNKT cells for many purposes, these iNKT cell lines should provide an important tool for the study of iNKT cell subsets, antigen and TCR specificity, activation, inactivation and effector functions.

Relationship between T-cell receptor α gene polymorphisms and symptomatic differences in patients with narcolepsy type 1.

BACKGROUND: Recent genome-wide association studies have identified an important role of T-cell receptor α (TRA) gene in the development of narcolepsy type 1. However, the role of TRA haplotype polymorphisms in the symptomatic diversity of narcolepsy remains unclear. This study aimed to investigate whether TRA polymorphisms can influence the symptomatic diversity of narcolepsy. METHODS: Totally, 903 patients with narcolepsy type 1 were included in the study. Patients were divided into different groups according to their symptoms. First, 13 genotyped single nucleotide polymorphisms in the TRA were assessed for their association with symptoms of narcolepsy. We used the Chi-square test to determine differences in genotype frequencies in patients with narcolepsy. Further, we identified the haplotypes and variations of the TRA and tested their association with the symptoms of narcolepsy using a logistic regression model. RESULTS: According to the results of the logistic regression, TRA haplotypes TG and CT were significantly associated with auditory hallucination, with odds ratios of 1.235 (95% confidence interval [CI], 1.012-1.507) and 1.236 (95% CI, 1.012-1.511), respectively (P < 0.05). CONCLUSIONS: The patterns of haplotype in TRA (haplotypes TG and CT) are associated with hypnagogic auditory hallucination in patients with narcolepsy type 1. However, further studies are needed to confirm our results and explore the underlying mechanisms.

Gene editing enables T-cell engineering to redirect antigen specificity for potent tumor rejection.

Adoptive transfer of TCR transgenic T cells holds great promise for treating various cancers. So far, mainly semi-randomly integrating vectors have been used to genetically modify T cells. These carry the risk of insertional mutagenesis, and the sole addition of an exogenous TCR potentially results in the mispairing of TCR chains with endogenous ones. Established approaches using nonviral vectors, such as transposons, already reduce the risk of insertional mutagenesis but have not accomplished site-specific integration. Here, we used CRISPR-Cas9 RNPs and adeno-associated virus 6 for gene targeting to deliver an engineered TCR gene specifically to the TCR alpha constant locus, thus placing it under endogenous transcriptional control. Our data demonstrate that this approach replaces the endogenous TCR, functionally redirects the edited T cells' specificity in vitro, and facilitates potent tumor rejection in an in vivo xenograft model.

Striking antigen recognition diversity in the Atlantic salmon T-cell receptor alpha/delta locus.

The complete TCR alpha/delta locus of Atlantic salmon (Salmo salar) has been characterized and annotated. In the 900 kb TCR alpha/delta locus, 292 Valpha/delta segments and 123 Jalpha/delta segments were identified. Of these, 128 Valpha/delta, 113 Jalpha, and a Jdelta segment appeared to be functional as they lacked frame shifts or stop codons. This represents the largest repertoire of Valpha/delta and Jalpha segments of any organism to date. The 128 functional Valpha/delta segments could be grouped into 29 subgroups based upon 70% nucleotide similarity. Expression data confirmed the usage of the diverse repertoire found at the genomic level. At least 99 Valpha, 13 Vdelta 86 Jalpha, 1 Jdelta, and 2 Ddelta segments were used in TCR alpha or delta transcription, and 652 unique genes were identified from a sample of 759 TCRalpha cDNA clones. Cumulatively, the genomic and expression data suggest that the Atlantic salmon T-cell receptor has enormous capacity to recognize a wide diversity of antigens.