CD3 Delta Chains Research Articles

Overcoming biases in causal inference of molecular interactions.

Computer inference of biological mechanisms is increasingly approachable due to dynamically rich data sources such as single-cell genomics. Inferred molecular interactions can prioritize hypotheses for wet-lab experiments to expedite biological discovery. However, complex data often come with unwanted biological or technical variations, exposing biases over marginal distribution and sample size in current methods to favor spurious causal relationships. Considering function direction and strength as evidence for causality, we present an adapted functional chi-squared test (AdpFunChisq) that rewards functional patterns over non-functional or independent patterns. On synthetic and three biology datasets, we demonstrate the advantages of AdpFunChisq over 10 methods on overcoming biases that give rise to wide fluctuations in the performance of alternative approaches. On single-cell multiomics data of multiple phenotype acute leukemia, we found that the T-cell surface glycoprotein CD3 delta chain may causally mediate specific genes in the viral carcinogenesis pathway. Using the causality-by-functionality principle, AdpFunChisq offers a viable option for robust causal inference in dynamical systems. The AdpFunChisq test is implemented in the R package 'FunChisq' (2.5.2 or above) at https://cran.r-project.org/package=FunChisq. All other source code along with pre-processed data is available at Code Ocean https://doi.org/10.24433/CO.2907738.v1. Supplementary materials are available at Bioinformatics online.

The NLPHL Tumor Microenvironment Is Markedly Enriched in the Tigit and PD-1 Signalling Axes Compared to Classical Hodgkin Lymphoma

The NLPHL Tumor Microenvironment Is Markedly Enriched in the Tigit and PD-1 Signalling Axes Compared to Classical Hodgkin Lymphoma

Variable presentation of primary immune deficiency: Two cases with CD3 gamma deficiency presenting with only autoimmunity

CD3 chain expression defects including CD3 gamma, epsilon, delta, and zeta chain subunits, are autosomal recessive inherited severe combined immunodeficiencies (SCID). The phenotype is usually T-B+NK+ SCID with lymphopenia where the clinical findings may be mild (CD3γ) or severe (CD3δ, ε, ζ) owing to the underlying molecular defect. There is limited information about the disease in literature. Here, we present two siblings from non-consanguineous family with autoimmunity including Evans syndrome, autoimmune hepatitis, nephrotic syndrome, and Hashimoto's thyroiditis and with no previous history of infections. To define the molecular basis of the disease, we performed linkage analysis around the CD3 receptor cluster and found consistent linkage to this region. The patient one displayed low TCRαβ expression, low IgG, low IgA, low IgM, low CD3, low CD4, low CD8. The patient two also displayed low TCRαβ expression and low anti-HBs titer. We went onto identify a homozygous splicing mutation (IVS2-1G>C) in the two affected individuals in the CD3γ gene. To date, only four cases have been reported with CD3γ deficiency. Occasionally, the patients present with only autoimmunity including autoimmune hemolytic anemia, vitiligo, Hashimoto's thyroiditis, and autoimmune enteropathy. However, Evans syndrome, autoimmune hepatitis, and nephrotic syndrome have not been reported in previous cases. We believe that our cases will contribute to the literature.

Hematopoietic stem cell transplantation in a CD3γ‐deficient infant with inflammatory bowel disease

Partial or total CD3 chain expression defects including CD3 gamma, epsilon, delta, and zeta chain are among the autosomally inherited SCID presenting with T-B+NK+ phenotype with lymphopenia. The clinical findings are generally severe in all except for CD3 gamma deficiency. Here we present a 10-month-old CD3 gamma deficient boy with IBD. The patient had suffered from intractable diarrhea, recurrent pulmonary infections and oral moniliasis since two months of age. Following the first allogeneic HSCT from his HLA-identical (6/6) sister after a reduced intensity regimen, a second transplantation was performed five months later. On day +19 after second transplantation, the CD3 TCR alpha/beta chain expression increased to 66% with development of full donor chimerism (98.6%). A significant improvement in diarrhea, perianal lesions, and rectal fistula was observed suggesting an improvement in inflammatory bowel disease. The patient died at home on day +50 with a sudden respiratory failure secondary to an undetermined infection. The case was interesting being the first reported case with SCID and inflammatory bowel disease who responded very well to HSCT by full recovery of intractable diarrhea, failure to thrive, laboratory findings, and improvement of fistula formation.

Biochemical Evidence for the Presence of a Single CD3δ and CD3γ Chain in the Surface T Cell Receptor/CD3 Complex

The T cell antigen receptor (TCR) consists of an alphabeta heterodimer and associated invariant CD3gamma, delta, epsilon, and zeta chains (TCR/CD3 complex). The general stoichiometry of the receptor complex, which is believed to be one molecule each of TCRalpha, TCRbeta, CD3gamma, and CD3delta and two molecules each of CD3epsilon and CD3zeta, is not clearly understood. Although it has been shown that there are two chains of CD3epsilon and CD3zeta, the stoichiometry of CD3gamma or CD3delta chains in the surface antigen receptor complex has not been determined. In the present study, transgenic mice expressing an altered form of mouse CD3delta and CD3gamma were employed to show that the surface TCR complexes contain one molecule each of CD3delta and CD3gamma. Thymocytes from wild type and CD3 chain transgenic mice on the appropriate knockout background were surface-biotinylated and immunoprecipitated using a specific antibody. The immunoprecipitates were resolved in two dimensions under nonreducing/reducing conditions to determine the stoichiometry of CD3delta and CD3gamma in the surface antigen receptor complex. Our data clearly show the presence of one molecule each of CD3delta and CD3gamma in the surface TCR/CD3 complex.

Expression and characterization of recombinant soluble human CD3 molecules: presentation of antigenic epitopes defined on the native TCR-CD3 complex.

The TCR-CD3 complex consists of the clonotypic disulfide-linked TCRalphabeta or TCRdeltagamma heterodimers, and the invariant CD3delta, epsilon, gamma and zeta chains. We generated plasmid constructs expressing the extracellular domains of the CD3delta, epsilon or gamma subunits fused to human IgG1 Fc. Recombinant fusion proteins consisting of individual CD3delta, epsilon or gamma subunits reacted poorly with anti-CD3 mAb including G19-4, BC3, OKT3 and 64.1. Co-expression of the CD3epsilon-Ig with either the CD3delta-Ig (CD3epsilondelta-Ig) or the CD3gamma-Ig (CD3epsilongamma-Ig) resulted in fusion proteins with much increased binding to G19-4. A brief acid treatment of the purified CD3epsilondelta-Ig fusion protein substantially improved its binding to BC3, OKT3 and 64.1. Surface plasmon resonance analysis revealed that the dissociation constants for CD3epsilondelta-Ig and anti-CD3 mAb ranged from 10(-8) to 10(-9) M. Based on these results, a single-chain (sc) construct encoding the CD3delta chain linked to the CD3epsilon chain with a flexible linker followed by human IgG1 Fc was expressed. The sc CD3deltaepsilon-scIg reacted with anti-CD3 mAb without requiring acid treatment. Moreover, anti-CD3 mAb bound CD3epsilondelta-Ig at a higher affinity than CD3epsilongamma-Ig, suggesting potential structural differences between the CD3epsilondelta and CD3epsilongamma subunits. In summary, we report the expression of soluble recombinant CD3 proteins that demonstrate structural characteristics of the native CD3 complex expressed on the T cell surface. These CD3 fusion proteins can be used to further analyze the structure of the TCR-CD3 complex, and to identify molecules that can interfere with TCR-CD3-mediated signal transduction by disrupting the interaction between CD3 and TCR subunits.

Some hints concerning the shape of T-cell receptor structures.

Several models are proposed for T-cell antigen receptor (TCR) assembly and structure. However, there is little experimental data favouring directly either one or the other(s). The minimal complex appears to be composed of a TCRalphabeta/CD3deltaepsilon,gammaepsilon/zeta2 structure but at the cell membrane, multimers of this minimal structure may be formed. Quantitative cytofluometry has suggested three CD3epsilon chains for two TCRbeta (or TCRdelta) chains/complex. Such data should be repeated with monoclonal antibodies (MoAb) against extracellular (EC) parts of CD3delta or CD3gamma chains. In the present review, we have compared the TCR/CD3 assembly of pre-TCR, TCRgammadelta and TCRalphabeta containing complexes, and analysed the reactivity of antibodies (Abs) against the EC part of CD3delta chains. Our data suggest an alternative assembly pathway and structure of TCR/CD3 complexes.

Evolution of T cell receptor (TCR) alpha beta heterodimer assembly with the CD3 complex.

T cell antigen receptors (TCR) are composed of an antigen-recognizing unit, the TCRalpha beta heterodimer, and a signal transduction ensemble, the CD3 complex. Whereas mammals possess three CD3 dimers (delta epsilon, gamma epsilon, and zeta2), birds and amphibians have only two (delta/gamma-epsilon and zeta2). To understand evolutionary changes in TCR/CD3 assembly,a phylogenetic approach was employed to dissect the interaction of TCRalpha beta heterodimers with the CD3 components. While sheep and mouse TCRalpha and TCRbeta chains could replace the corresponding human chains in mutant human T cells to restore surface TCR/CD3 expression and function, chicken TCRalpha, TCRbeta and CD3delta/gamma chains were unable to replace the corresponding human chains in forming a chimeric TCR/CD3 complex. The inability of chicken TCR/CD3 components to replace the human molecules in T cells was found to result from the lack of interaction between chicken TCRalpha beta heterodimers and the human CD3 complex. In contrast, if no CD3 molecules are present (non-T cells), TCRalpha -TCRbeta chain pairing can take place in an apparently non-controlled way. Thus, the TCR-CD3 interactions have changed with the evolutionary divergence of two mammalian CD3gamma and CD3delta genes from a single prototypic chicken delta/gamma gene. Our data suggest that the structures in mammalian TCR.C regions, which distinguish between CD3delta and CD3gamma chains, have evolved with the appearance of two separate CD3delta and CD3gamma functions.

On the role of CD3delta chains in TCRgammadelta/CD3 complexes during assembly and membrane expression.

The present study was performed in order to analyze whether T-cell receptor (TCR)/CD3 assembly, intracellular transport and surface expression are carried in a similar way in alphabeta-and gammadelta-T cells. By means of optimal immunoprecipitation conditions with 35S-methionine/cysteine- or biotin-labelled TCR/CD3 proteins from alphabeta- or gammadelta-T-lymphoma-cell lines, as well as TCRgammadelta cDNA transfectants, it was found that CD3delta chains associate less strongly with TCRgammadelta heterodimers compared to TCRalphabeta heterodimers. This preferential reactivity of CD3delta chains appears to be structural and not owing to differences in gammadelta- versus alphabeta-T-cell intracellular environments. Our results are in accordance firstly, with data from CD3delta-deficient mice, which have gammadelta-T cells but no alphabeta-T cells, secondly with the suggested role of CD3delta chains in the positive selection of alphabeta-T cells, a process apparently not followed by gammadelta-T cells, and lastly with the differential roles of CD3delta chains versus CD3gamma chains, explaining the maintenance of two CD3delta and CD3gamma genes after the duplication from a CD3delta/gamma gene present in avians. The impaired reactivity of CD3delta chains with TCRgammadelta heterodimers seems to be owing to a less efficient association with TCRgamma chains. In contrast, CD3delta chains interact as strongly with TCRdelta chains as do CD3gamma chains with both TCRgamma and TCRdelta chains. These data may explain, at the molecular levels, why surface TCR/CD3 expression levels are impaired in gammadelta-T cells from CD3gamma-deficient mice but not from CD3delta-deficient mice.

CD3 polymorphism in cynomolgus monkeys (Macaca fascicularis).

Cynomolgus monkeys were divided into two groups in terms of the reactivity of their lymphocytes with the FN18 monoclonal antibody, which is directed to the CD3 of rhesus monkeys. It was shown that 24 (12.2%) out of 196 monkeys did not have lymphocytes that reacted with the FN18, although T cells from those animals responded well to mitogenic stimulation. We have determined the nucleotide sequences of the CD3delta, CD3gamma, and CD3epsilon chains and found that two amino acids of the CD3epsilon chain of the FN18 non-reactive monkeys were different when compared with the FN18 reactive monkeys. Our results indicated that the CD3epsilon molecule of cynomolgus monkeys is polymorphic at the epitope level, which is recognized by the FN18 monoclonal antibody.

CD3delta couples T-cell receptor signalling to ERK activation and thymocyte positive selection.

Thymocytes from mice lacking the CD3delta chain of the T-cell receptor (TCR), unlike those of other CD3-deficient mice, progress from a CD4- CD8- double-negative to a CD4+ CD8+ double-positive stage. However, CD3delta-/- double-positive cells fail to undergo positive selection, by which double-positive cells differentiate into more mature thymocytes. Positive selection is also impaired in mice expressing inactive components of the Ras/mitogen activated protein (MAP) kinase signalling pathway. Here we show that CD3delta-/- thymocytes are defective in the induction of extracellular signal-regulated protein kinase (ERK) MAP kinases upon TCR engagement, whereas activation of other MAP kinases is unaffected. The requirement for CD3delta maps to its extracellular or transmembrane domains, or both, as expression of a tail-less CD3delta rescues both ERK activation and positive selection in CD3delta-/- mice. Furthermore, the defect correlates with severely impaired tyrosine phosphorylation of the linker protein LAT, and of the CD3zeta chain that is localized to membrane lipid rafts upon TCR engagement. Our data indicate that the blockade of positive selection of CD3delta-/- thymocytes may derive from defective tyrosine phosphorylation of CD3zeta in lipid rafts, resulting in impaired activation of the LAT/Ras/ERK pathway.

An amphibian CD3 homologue of the mammalian CD3 gamma and delta genes.

T cell receptor (TCR) genes have been identified in representatives of both cartilaginous and bony vertebrates. The CD3 chains that serve as signal transducing elements of the TCR complex in mammals have been defined to a limited extent in birds. In these studies a CD3 homologue was identified in an amphibian representative, Xenopus laevis, using degenerate oligomer primers designed from conserved regions of avian and mammalian CD3 gamma/delta subunits. The reverse transcriptase polymerase chain reaction amplified product of Xenopus splenocyte RNA was then used to isolate full-length cDNA clones from a splenic library. When employed as probes, the cDNA clones hybridized with a 1-kb mRNA transcript in Xenopus T cells, but not in other cell types. Comparison of the deduced amino acid sequence indicated a similar degree of homology with mammalian and avian CD3 gamma and delta chains. Genomic analysis indicated that the Xenopus CD3 molecule is encoded by five exons, a structure resembling the mammalian CD3 delta gene rather than the seven exon CD3 gamma gene. Southern blot analysis and sequencing of the 5' flanking region failed to yield evidence of a related Xenopus gene. This amphibian CD3 gene thus appears to represent an ancestral form of the mammalian CD3 gamma and delta genes.

CD3 delta deficiency arrests development of the alpha beta but not the gamma delta T cell lineage.

The CD3 complex found associated with the T cell receptor (TCR) is essential for signal transduction following TCR engagement. During T cell development, TCR-mediated signalling promotes the transition from one developmental stage to the next and controls whether a thymocyte undergoes positive or negative selection. The roles of particular CD3 components in these events remain unclear. Indeed, it is unknown whether they have specialized or overlapping roles. However, the multiplicity of CD3 components and their evolutionary conservation suggest that they serve distinct functions. Here the developmental requirement for the CD3 delta chain is analyzed by generating a mouse line specifically lacking this component (delta-/- mice). Strikingly, CD3 delta is shown to be differentially required during development. In particular, CD3 delta is not needed for steps in development mediated by pre-TCR or gamma delta TCR, but is required for further development of thymocytes expressing alpha beta TCR. Absence of CD3 delta specifically blocks the thymic selection processes that mediate the transition from the double-positive to single-positive stages of development.

Qualitative and quantitative contributions of the T cell receptor zeta chain to mature T cell apoptosis.

Engagement of the T cell receptor (TCR) of mature T lymphocytes can lead either to activation/proliferation responses or programmed cell death. To understand the molecular regulation of these two fundamentally different outcomes of TCR signaling, we investigated the participation of various components of the TCR-CD3 complex. We found that the TCR-zeta chain, while not absolutely required, was especially effective at promoting mature T cell apoptosis compared with the CD3 epsilon, gamma, or delta chains. We also carried out mutagenesis to address the role of the immunoreceptor tyrosine-based activation motifs (ITAMs) that are the principal signaling components found three times in the TCR-zeta chain and once in each of the CD3 epsilon, gamma, or delta chains. We found that the ability of the TCR-zeta chain to promote apoptosis results both from a quantitative effect of the presence of multiple ITAMs as well as qualitatively different contributions made by individual ITAMs. Apoptosis induced by single chain chimeras revealed that the first zeta ITAM stimulated greater apoptosis than the third zeta ITAM, and the second zeta ITAM was unable to trigger apoptosis. Because microheterogeneity in the amino acid sequence of the various ITAM motifs found in the TCR-zeta and CD3 chains predicts interactions with distinct src-homology-2-domain signaling proteins, our results suggest the possibility that individual ITAM motifs might play unique roles in TCR responses by engaging specific signaling pathways.

Composition of TCR-CD3 complex in human intestinal intraepithelial lymphocytes: lack of FcεRlγ chain

Human intestinal intraepithelial lymphocytes (iIEL) are a unique population of predominantly CD8 alpha beta+, TCR alpha beta+ lymphocytes and, to a lesser extent, TCP gamma delta+ lymphocytes that proliferate poorly to anti-CD3 mitogenic signals but display significant cytolytic activity. Studies in mouse model systems have shown that the gamma chain of the high-affinity receptor for IgE (Fc epsilon RI gamma) may substitute for the zeta chain in the TCR-CD3 complex of iIEL. This has suggested that the functional properties of these cells may be associated with an altered composition of the TCR-CD3 complex. We therefore analyzed the TCR-CD3 complex of normal human iIEL. One- and two-dimensional non-reducing/reducing SDS-PAGE analysis of CD3 gamma, CD3 delta, CD3 epsilon, zeta and Fc epsilon RI gamma chain immunoprecipitates of cell surface radiolabeled proteins with subunit-specific antibodies revealed a TCR-CD3 complex without associated Fc epsilon RI gamma chains. Thus, normal human iIEL contain a TCR-CD3 complex that consists predominantly of zeta homodimers in association with the alpha beta TCR and CD3 gamma, delta and epsilon, similar to the majority of peripheral lymphocytes. This indicates that the distinct properties of human iIEL are not associated with substitutions of the Fc epsilon RI gamma chain in the TCR-CD3 complex.

T cell receptor expression by dendritic epidermal T cells.

Dendritic epidermal T cells (DETC) in murine epidermis express the gamma delta T cell receptor (TCR). The major population of DETC utilize V gamma 5 and V delta 1 without any junctional diversity, corresponding to the earliest fetal thymocytes which express TCR gamma delta. Using PCR, we recently found another population of DETC which express V gamma 1-V delta 6 with junctional diversity in addition to V gamma 5-V delta 1, although they exist in small numbers in normal mice. In athymic nude mice, V gamma 1+ cells also exist. Therefore, this subset of gamma delta T cells is the product of an extrathymic pathway. These V gamma 1+ cells may recognize mycobacterium antigen or heat shock protein (HSP), thus playing an important role in the first defense of the skin. In contrast to normal mice, in nude mice, we could not detect any DETC using anti CD3 epsilon antibody (2C11). In order to solve this puzzle, we examined the components of TCR complex utilizing immunoprecipitation and northern blot analysis. TCR is composed of either alpha beta or gamma delta chains associated with CD3 gamma, delta, epsilon and zeta-zeta chains. By immunoprecipitation of 125I labelled DETC cell lines using anti-CD3 epsilon antibody, we detected gamma delta chains and CD3 gamma and CD3 epsilon chains, but not CD3 delta or CD3 zeta chains. Northern blot analysis showed that these cells express CD3 gamma, epsilon, and zeta chains, but not the CD3 delta chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Ig heavy chain extracellular spacer confers unique glycosylation of the Mb-1 component of the B cell antigen receptor complex.

A unique functional role for the B cell Ag receptor, IgD, has not yet been identified. A number of properties of IgD, such as distinct intron-exon organization and a conserved pattern of developmental expression, suggest a selective evolutionary advantage for this receptor isotype. To explore structural features of IgD that may confer unique functions, chimeric Ag receptors were generated in which small segments of the IgD heavy chain membrane proximal regions were substituted for corresponding segments of the IgM heavy chain. Polypeptides that associate with the Ig receptors were analyzed. Mb-1/Ig-alpha, a signal transduction molecule, is known to be glycosylated in a distinct manner when associated with IgD compared to when associated with other isotypes. We report that this differential glycosylation results because one N-linked carbohydrate on Mb-1 fails to be processed into an Endo-H-resistant form when associated with IgM or IgG, whereas both N-linked carbohydrates are processed on Mb-1 associated with IgD. By preparing chimeric IgM-IgD heavy chains, substitution of the IgD extracellular spacer segment alone was found to be necessary and sufficient to confer an IgD-type glycosylation pattern on the Mb-1 molecule. This altered glycosylation pattern, however, did not confer a detectable difference in calcium mobilization or in protein tyrosine phosphorylation upon receptor stimulation. Interestingly, a similar altered glycosylation pattern has been reported for the T cell Ag receptor complex in which carbohydrates on the CD3 delta-chain are fully processed in gamma delta-T cell receptors but only partially processed in alpha beta-T cell receptors.

Association of Raf with the CD3 delta and gamma chains of the T cell receptor-CD3 complex.

Protein kinases and phosphatases play an important role in signal transduction. In the T cell, activation via the T cell receptor-CD3 complex results in rapid tyrosine phosphorylation of proteins, as well as subsequent increases in serine/threonine phosphorylation. The Raf serine/threonine kinase has been implicated in many receptor signaling pathways, including those of platelet-derived growth factor, epidermal growth factor, insulin, and interleukin-2 receptors. We show here that Raf is associated with the T cell receptor-CD3 complex in unstimulated murine T cells. Using a COS cell expression system, we show that a hypophosphorylated form of Raf specifically associates with the CD3 gamma and delta chains but not with the CD3 epsilon or zeta chains. These results suggest that Raf mediates signal transduction from the gamma and delta chains of the T cell receptor-CD3 complex, analogous to the role played by ZAP-70 in signal transduction mediated by the zeta chain.

CD3 epsilon-mediated signals rescue the development of CD4+CD8+ thymocytes in RAG-2-/- mice in the absence of TCR beta chain expression.

Recent studies have shown that TCR beta chain expression can effect the differentiation of CD4-CD8- double-negative (DN) thymocytes to CD4+CD8+ double-positive (DP) thymocytes. The TCR beta chain is expressed on the surface of DP thymocytes in association with CD3 gamma, delta and epsilon chains, suggesting a potential role for CD3 components in this signaling process. We now report detection of a very low level of surface expression of CD3 epsilon on adult DN RAG-2-/- thymocytes. This surface CD3 epsilon was associated with CD3 gamma and delta chains, as detected by anti-CD3 epsilon immunoprecipitation analyses. Significantly, injection of anti-CD3 epsilon mAb into RAG-2-/- mice led to the accumulation of an IL-2R alpha- CD2+ DP cell population and a nearly 100-fold increase in thymic cellularity to essentially normal levels. Together, these data strongly indicate that TCR beta chain-mediated developmental signals are transduced by CD3 components and provide potential insights into mechanisms by which TCR beta chain expression may effect this process.

Specificity of signal transduction through CD16, TCR-CD3 and BCR receptor chains containing the tyrosine-associated activation motif.

FcR gamma III (CD16) is a member of a family of receptor proteins that bind to Fc regions of IgG molecules. Ligation of CD16 transduces signal and alters tyrosine phosphorylation of proteins in natural killer, B and T cells. However, the identity of protein tyrosine kinases (PTKs) involved in CD16 signaling and the mode of their interaction with the receptor are not completely understood. Here, we show that the transmembrane form of CD16 transfected in a CD3- Jurkat cell line associates with p56lck and ZAP-70 PTKs. Using chimeric proteins consisting of the extracellular domain of CD16 and the cytoplasmic portions of the zeta chain, we find that these PTKs interact with the 17-residue activation motif present in the zeta chain. Substitution of tyrosine residues in the motif with phenylalanine blocks the interaction of PTKs and abrogates signal transduction. Comparative studies demonstrate that similar to zeta, MB1 protein of the IgM receptor also binds to p56lck and ZAP-70 in T cells and induces strong activation responses, whereas CD3 gamma, delta and epsilon chains do not interact with ZAP-70 and transduce weak signals. Together, these results demonstrate that: (i) PTKs bound to CD16 mediate signal transduction, (ii) ZAP-70 plays a crucial role in signaling and (iii) apart from tyrosine residues in the activation motif, other structural constraints also regulate the interaction of PTKs with the receptor molecule.