CD3 Chains Research Articles

Critical and Multiple Roles for the CD3ε Intracytoplasmic Tail in Double Negative to Double Positive Thymocyte Differentiation

The preTCR is associated with signal-transducing CD3gamma, delta, epsilon, and zeta polypeptides. It is generally agreed that CD3 chains play redundant roles in the receptor-mediated signal transduction. In the present study, we show that the intracytoplasmic (IC) domain of CD3epsilon is essential for early thymocyte maturation. We demonstrate that the IC domain-deleted CD3epsilon fails to restore the double negative (DN) to double positive (DP) thymocyte development in CD3epsilon-deficient mice. Additional experiments show that the membrane proximal basic amino acid rich sequence in the IC domain of CD3epsilon is sufficient for the DN to DP differentiation, whereas the proline rich sequence is required for efficient proliferation. This is probably due to impaired ligand independent recruitment of Nck to the proline rich sequence motif of CD3epsilon within the context of the preTCR. The data presented in this study elucidates mechanistic basis for the preTCR-induced proliferation of the DN thymocytes and have identified distinct roles for individual motifs of CD3epsilon in the preTCR-mediated differentiation and proliferation. These data provide the first genetic and phenotypic evidence for requirement of the IC domain of a CD3 chain in thymocyte development.

A Biophysical Mechanosensor Model for T-Cell Receptor Signaling

T-cells (cytolytic, helper and others) are vital components in adaptive mammalian immune defense systems. T-cell receptor (TCR) signaling which follows recognition of a peptide antigen bound to an MHC molecule (pMHC) on an antigen presenting cell (APC) is critical for T-cell activation, differentiation, and proliferation. For decades, the mechanism for TCR signaling across the T-cell membrane by this multi-subunit receptor has remained a mystery. Based on our recently obtained structural data on stimulatory and non-stimulatory anti-TCR antibodies and their binding footprint to TCR components obtained by NMR, we present here a simple but all-inclusive “dynamic torque transducer” model for TCR signaling. Extracellular mechanical torque can result in quaternary structure changes between the pMHC-binding TCR alpha/beta chains and the non-convalently linked CD3 chains within the TCR complex, triggering downstream signaling via ITAMs in the CD3 cytoplasmic tails. In this process, the pMHC behaves as a detachable effort arm of this complex-lever system; its binding is determined by the specificity of an individual TCR but is not sufficient to mediate signal transduction per se. An external torque is additionally required to provide the energy for this signaling. Such a torque can be generated either via a shear force or vertical pressure between the opposing APC and T cell membranes. The former “bind-and-tug” mode may be responsible for initial signaling during T-cell scanning of APCs, while the latter “bind-and-bend” mode is responsible for sustained signaling inside immune-synapses during T-cell activation. Our model can also explain T-cell signaling mediated by antibody or multimeric pMHC cross-linking. This mechanism of converting mechanical energy to a biochemical signal, mediated and controlled by a detachable interface, may be generally applicable in other cell-cell signaling systems both within and outside of the immune system.

Clinical, histopathological and immunohistological study of lymphoid disorders in the parotid gland of patients with Sjögren's syndrome

Sjögren's syndrome is a chronic autoimmune systemic disease characterized by polyglandular tissue destruction, leading to keratoconjunctivitis sicca and xerostomia. These patients have 44-fold increased risk of developing salivary gland lymphoma, of which 80% are marginal zone (MALT) type. Having in mind that criteria for distinguishing benign lymphoepithelial lesions from MALT lymphoma are obscure, the aim of this study was to provide practical information that could be integrated into diagnostic practice. Among 32 parotidectomies, 27 cases were identified as having benign lymphoepithelial disorders and 5 cases low grade MALT lymphoma. Histological sections were stained routinely with hematoxylin and eosin (H&E and special stains. Immunohistochemical study was performed by LSAB2 method, by using primary antibodies for CD20, CD3, Kappa and Lambda light chains and Cytokeratin (Dako Denmark). The 27 patients with Sjögren's sialoadenitis (22 women and 5 men), and 5 patients with MALT lymphoma (only women) were included in this analysis. According to the Ann Harbor Classification, all patients with MALT lymphoma had stage IE. Both groups of patients had an indolent clinical course, except permanent, rapid parotid enlargement in the patients with MALT lymphoma. Histologically, the periductal lymphoid infiltrate, gradually extended to the acini, completely replacing them by a sea of polyclonal lymphocytes, immunoblasts, germinal centers and plasma cells (confirmed immunohistochemically), but sparing the ducts and preserving lobular appearance. The histological feature of salivary gland MALT lymphoma included heterogeneous B-cell infiltrate that totally or subtotally had effaced the normal glandular structure. Malign lymphoepithelial lesions, representing infiltration of the ductal and epithelial structures by monoclonal neoplastic B-cells, positive for CD20, were highlighted by antibody to cytokeratin. The optimal diagnosis of salivary gland MALT lymphoma requires careful integration of clinical, morphological and immunohistochemical results.

Mantle cell lymphoma with flow cytometric evidence of clonal plasmacytic differentiation: A case report

Plasmacytic differentiation in mantle cell lymphoma (MCL) occurs rarely. However, no flow cytometric studies that demonstrate plasmacytic (PC) differentiation in MCL have been reported. Herein, we report a case of MCL with PC differentiation identified by flow cytometry. Morphologic review was performed by hematoxylin and eosin (H&E) stained sections from paraffin-embedded lymph node, colon and bone marrow specimens, and Wright-Geimsa stained bone marrow aspirate smears and touch imprints. Immunohistochemical stains using antibodies against CD3, CD5, CD20, and cyclin-D1, and in-situ hybridization for kappa and lambda light chains were reviewed. Multicolor flow cytometry analysis was performed on the bone marrow aspirate with monoclonal antibodies to CD3, CD4, CD5, CD8, CD14, CD19, CD20, CD23, CD38, CD45, CD56, CD138, and kappa and lambda light chains. FISH analysis for t(11;14)(q13;q32) was performed on interphase cells. The neoplastic cells had the cytologic features of MCL with nodal, bone marrow, and colonic involvement. In-situ hybridization for kappa and lambda light chains demonstrated clonal plasma cells in the lymph node and bone marrow biopsies. In addition, flow cytometric studies of the bone marrow aspirate showed three populations of neoplastic cells: a clonal B-cell population with typical MCL phenotype, a similar B-cell population in transition to plasma cells, and a clonal plasma cell population. The plasma cells retained CD5 expression and had the same light chain restriction as the clonal B-cells. Multi-parameter flow cytometry can be useful in demonstrating clonal PC differentiation in MCL and distinguishing from a concurrent but unrelated plasma cell dyscrasia.

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.

Recombinase‐deficient T cell development by selective accumulation of CD3 into lipid rafts

The pre-T cell receptor (pre-TCR) promotes the development of thymocytes with productive rearrangement at the TCR beta chain locus by signaling in a ligand-independent fashion. The TCR beta chain associates with the invariant pre-Talpha (pTalpha) chain, which bears specific charged residues in the extracellular portion mediating pre-TCR self-oligomerization. In recombinase-deficient thymocytes, calnexin (CNX) associated with CD3 chains is inefficiently retained in the endoplasmic reticulum (ER) and weakly expressed in the plasma membrane. Deliberate cross-linking of CNX/CD3 complexes mimics pre-TCR signaling. Here, we show that, analogously to the pTalpha chain, surface CNX is palmitoylated and that CD3 prominently accumulated in lipid rafts upon cross-linking. Mutant CNX isoforms devoid of ER retention determined pre-TCR-like signaling and simulated beta selection only when stably translocating CD3 to lipid rafts. Inclusion of the palmitoylated cytoplasmic tail from the pTalpha chain in recombinant CNX strikingly improved the pre-TCR-like signaling efficiency of CNX/CD3 in rafts. This study indicates that lipid rafts in the plasma membrane represent proficient microdomains for the initiation of pre-TCR signaling, and supports the view that beta selection by oligomerized pre-TCR is implemented by the pTalpha cytoplasmic tail.

Hypothesis: TCR signal transduction—A novel tri-modular signaling system

Antigenic peptides initiate an immune response in T cells via the T cell receptor (TCR). The TCR itself is widely regarded as one of the most complex receptors in nature, as it is comprised of at least six different subunits, the antigen recognizing TCRα and β chains, and the signal transmitting CD3δɛ, γɛ, and ζ 2 dimers. In order for a signal to be transmitted from the TCR to the cytoplasm, the CD3 chains must “sense” that an antigenic peptide has been presented to the TCRα and β subunits. After accomplishing this, there are a total of 10 different immunoreceptor tyrosine activation motifs (ITAMs) present within the CD3 chains which effectively activate the T cell and hence the immune response. The importance of each CD3 chain and subsequently each ITAM has been the focus of intense research. However, the precise role(s) played by each CD3 chain has remained elusive. Using the immunomodulatory peptide termed core peptide (CP), which is proposed to inhibit TCR activation by disrupting TCR–CD3 interactions, a tri-modular signaling system for T cell activation is proposed. By contrast to the existing two distinct signaling model (ζ 2, CD3ɛγ/ɛδ), in this model each of the three dimers, CD3γɛ, δɛ, and ζ 2, are proposed to act as three separate and distinct signaling modules, performing both specific and redundant functions.

Altered T-Cell Function in Schizophrenia: A Cellular Model to Investigate Molecular Disease Mechanisms

Despite decades of research into the aetiology and pathophysiology of schizophrenia, our understanding of this devastating disorder remains incomplete, with adverse consequences for both diagnosis and treatment. Here we investigate whether differences between patients and controls can be observed in peripheral patient tissue, with a view of establishing a means for dynamic investigations into cell function. In vitro stimulation of peripheral blood CD3+ pan T cells with anti-CD3 (clone OKT3) was used to investigate disease-associated cell responses. T cells from both medicated (n = 39), unmedicated (n = 6) and minimally medicated (n = 5) schizophrenia patients were found to have significantly lower proliferative responses to stimulation, compared to well-matched controls (n = 32). Expression of CD3 and TCR (T cell receptor) αβ chains was equivalent between patients and controls, ensuring equal stimulation with anti-CD3, and there was no significant difference in the proportions of CD4+ and CD8+ T cells between samples (n = 12). Lower T cell proliferation in schizophrenia patients was not found to result from deficient early tyrosine phosphorylation signalling or lower IL-2 (interleukin-2) production, as these parameters were similar between patients and controls, as was the expression of CD25, the IL-2 receptor α chain. Analysis of CD45 isoforms, however, revealed that patients had a significantly greater percentage of CD8+ and CD4+ CD45RA+ cells before stimulation and significantly higher fluorescence intensity of CD45RA on CD4+ and CD8+ cells before and after stimulation. There was significantly higher expression of CD45 RB on both CD4+ and CD8+ unstimulated cells, with a trend towards lower numbers of CD45RO+ T cells in patient blood. Gene expression analysis in freshly isolated T cells from six minimally treated or first onset patients and six controls was carried out using human whole-genome CodeLink microarrays to identify functional pathways that may affect the ability of patient cells to respond to stimulation. Functional profiling showed prominent transcript changes in categories pertaining to cell cycle machinery, intracellular signalling, oxidative stress and metabolism. Intriguingly, chromosomal location analysis of genes significantly altered between schizophrenia and controls revealed clusters at 1p36, 1q42 and 6p22, which have previously been identified as strong susceptibility loci for schizophrenia.

CD8 Raft Localization Is Induced by Its Assembly into CD8αβ Heterodimers, Not CD8αα Homodimers

The coreceptor CD8 is expressed as a CD8alphabeta heterodimer on major histocompatibility complex class I-restricted TCRalphabeta T cells, and as a CD8alphaalpha homodimer on subsets of memory T cells, intraepithelial lymphocytes, natural killer cells, and dendritic cells. Although the role of CD8alphaalpha is not well understood, it is increasingly clear that this protein is not a functional homologue of CD8alphabeta. On major histocompatibility complex class I-restricted T cells, CD8alphabeta is a more efficient TCR coreceptor than CD8alphaalpha. This property has for the mouse protein been attributed to the recruitment of CD8alphabeta into lipid rafts, which is dependent on CD8beta palmitoylation. Here, these divergent distributions of CD8alphabeta and CD8alphaalpha are demonstrated for the human CD8 proteins as well. However, although palmitoylation of both CD8alpha and CD8beta chains was detected, this modification did not contribute to raft localization. In contrast, arginines in the cytoplasmic domain are crucial for raft localization of CD8betabeta. Most strikingly, the assembly of a non-raft localized CD8beta chain with a non-raft localized CD8alpha chain resulted in raft-localized CD8alphabeta heterodimers. Using chimeric CD8 proteins, this property of the heterodimer was found to be determined by the assembly of CD8alpha and CD8beta extracellular regions. The presence of two CD8alpha extracellular regions, on the other hand, appears to preclude raft localization. Thus, heterodimer formation and raft association are intimately linked for CD8alphabeta. These results emphasize that lipid raft localization is a key feature of human CD8alphabeta that clearly distinguishes it from CD8alphaalpha.

Plasmablastic lymphoma of visceral cranium, cervix and thorax in an HIV-negative woman

Dear Editor, We would like to report a case of aggressive plasmablastic lymphoma (PBL) in an HIV-negative individual, characterised by a unique combination of unusual clinical and immunophenotypic features. A 67-year-old woman presented with a 20-day history of a painless mass lesion in the left supraclavicular fossa. She was also complaining of blurred vision and a continuous mild retrosternal pain. She denied fever, night sweats or weight loss. Her past medical history was negative for diseases or treatments associated with immunodeficiency. On physical examination, she had diplopia on upward gaze. A painless firm mass 3×4 cm in size was palpated in the left supraclavicular fossa. There was no peripheral lymphadenopathy, organomegaly or other remarkable findings. Laboratory investigations showed a normal full blood count and serum biochemical profile, including normal lactate dehydrogenase levels. No paraprotein was present on serum and urine electrophoresis. Enzyme-linked immunosorbent assay testing for HIV antibodies was negative. Computerised tomography (CT) imaging studies revealed a mass with soft tissue density occupying the left sphenoid sinus and infiltrating the ethmoid sinus and ipsilateral concha with associated areas of bone destruction. The mass was extending to the left orbit with thickening of the left inferior rectus muscle. There was also an evidence of tumour invasion of the left maxillary antrum and mandibular ramus. Furthermore, soft tissue masses compatible with multiple lymph node enlargement and block formation were present in the inferior frontolateral cervical region with extension to the anterior upper mediastinum. CT imaging of abdomen was negative for lymphadenopathy or organomegaly. A bone marrow biopsy was negative for neoplastic invasion. Biopsy of the cervical mass revealed a neoplasm causing complete effacement of nodal architecture and extending to the adjacent adipose tissue. The neoplastic tissue consisted of diffusely growing largeand medium-sized cells with polymorphic nuclei, frequent mitoses and moderate-to-large amount of eosinophilic or amphiphilic cytoplasm (Fig. 2a). Immunohistochemical investigation using markers of epithelial (Ker HMW, Ker LMW, Ker7, Ker20, EMA, Cam 5-2), lymphoid (CD3, CD20, CD45RO, CD45, CD30, CD21, CD79a, CD8, CD56, CD138, kappa and lambda chains, immunoglobulins), mesenchymal (S-100, vimentin, actin, desmin, CD31, CD34, GFAP (glial fibrillary acidic protein)), histiocytic (CD68), melanocytic (HMB-45) differentiation and various others (Bcl-2, CD99, neuron-specific enolase [NSE]) revealed only a strong expression of vimentin and a small percentage of cells with moderate positivity for NSE. Blind biopsies from the nasopharynx indicated only nonspecific immunologic stimulation of the lymphoid tissue. Ann Hematol (2007) 86:615–618 DOI 10.1007/s00277-007-0280-z

Differential Biological Role of CD3 Chains Revealed by Human Immunodeficiencies

The biological role in vivo of the homologous CD3gamma and delta invariant chains within the human TCR/CD3 complex is a matter of debate, as murine models do not recapitulate human immunodeficiencies. We have characterized, in a Turkish family, two new patients with complete CD3gamma deficiency and SCID symptoms and compared them with three CD3gamma-deficient individuals belonging to two families from Turkey and Spain. All tested patients shared similar immunological features such as a partial TCR/CD3 expression defect, mild alphabeta and gammadelta T lymphocytopenia, poor in vitro proliferative responses to Ags and mitogens at diagnosis, and very low TCR rearrangement excision circles and CD45RA(+) alphabeta T cells. However, intrafamilial and interfamilial clinical variability was observed in patients carrying the same CD3G mutations. Two reached the second or third decade in healthy conditions, whereas the other three showed lethal SCID features with enteropathy early in life. In contrast, all reported human complete CD3delta (or CD3epsilon) deficiencies are in infants with life-threatening SCID and very severe alphabeta and gammadelta T lymphocytopenia. Thus, the peripheral T lymphocyte pool was comparatively well preserved in human CD3gamma deficiencies despite poor thymus output or clinical outcome. We propose a CD3delta >> CD3gamma hierarchy for the relative impact of their absence on the signaling for T cell production in humans.

A Membrane-proximal Tetracysteine Motif Contributes to Assembly of CD3δϵ and CD3γϵ Dimers with the T Cell Receptor

Assembly of the T cell receptor (TCR) with its dimeric signaling modules, CD3deltaepsilon, CD3gammaepsilon, and zetazeta, is organized by transmembrane (TM) interactions. Each of the three assembly steps requires formation of a three-helix interface involving one particular basic TCR TM residue and two acidic TM residues of the respective signaling dimer. The extracellular domains of CD3deltaepsilon and CD3gammaepsilon contribute to assembly, but TCR interaction sites on CD3 dimers have not been defined. The structures of the extracellular domains of CD3deltaepsilon and CD3gammaepsilon demonstrated parallel beta-strands ending at the first cysteine in the CXXCXEXXX motif present in the stalk segment of each CD3 chain. Mutation of the membrane-proximal cysteines impaired assembly of either CD3 dimer with TCR, and little complex was isolated when all four membrane-proximal cysteines were mutated to alanine. These mutations had, however, no discernable effect on CD3deltaepsilon or CD3gammaepsilon dimerization. CD3deltaepsilon assembled with a TCRalpha mutant that lacked both immunoglobulin domains, but shortening of the TCRalpha connecting peptide reduced assembly, consistent with membrane-proximal TCRalpha-CD3deltaepsilon interactions. Chelation of divalent cations did not affect assembly, indicating that coordination of a cation by the tetracysteine motif was not required. The membrane-proximal cysteines were within close proximity but only formed covalent CD3 dimers when one cysteine was mutated. The four cysteines may thus form two intrachain disulfide bonds integral to the secondary structure of CD3 stalk regions. The three-chain interaction theme first established for the TM domains thus extends into the membrane-proximal domains of TCRalpha-CD3deltaepsilon and TCRbeta-CD3gammaepsilon.

PARP-2 deficiency affects the survival of CD4+CD8+ double-positive thymocytes

Poly-(ADP-ribose) polymerase-2 (PARP-2) belongs to a large family of enzymes that synthesize and transfer ADP-ribose polymers to acceptor proteins, modifying their functional properties. PARP-2-deficient (Parp-2-/-) cells, similar to Parp-1-/- cells, are sensitive to both ionizing radiation and alkylating agents. Here we show that inactivation of mouse Parp-2, but not Parp-1, produced a two-fold reduction in CD4+CD8+ double-positive (DP) thymocytes associated with decreased DP cell survival. Microarray analyses revealed increased expression of the proapoptotic Bcl-2 family member Noxa in Parp-2-/- DP thymocytes compared to littermate controls. In addition, DP thymocytes from Parp-2-/- have a reduced expression of T-cell receptor (TCR)alpha and a skewed repertoire of TCRalpha toward the 5' Jalpha segments. Our results show that in the absence of PARP-2, the survival of DP thymocytes undergoing TCRalpha recombination is compromised despite normal amounts of Bcl-xL. These data suggest a novel role for PARP-2 as an important mediator of T-cell survival during thymopoiesis by preventing the activation of DNA damage-dependent apoptotic response during the multiple rounds of TCRalpha rearrangements preceding a positively selected TCR.

Cytohesin Binder and Regulator Augments T Cell Receptor-induced Nuclear Factor of Activated T Cells·AP-1 Activation through Regulation of the JNK Pathway

Cytohesin binder and regulator (Cybr; also known as CYTIP, CASP, and PSCDBP) is a cytokine-induced gene preferentially expressed in hematopoietic tissues and in T helper 1 cells. Cybr protein associates with members of the cytohesin family, which are known ADP-ribosylation factors-GDP/GTP exchange factors, and its functions appear to regulate lymphocyte adhesion and cell-cell contact. Here we show that Cybr mRNA and protein levels are increased upon T cell receptor engagement. Cybr expression then influences T cell receptor-dependent signaling events, such as nuclear factor of activated T cells and AP-1 transcriptional activity. In addition, expression of Cybr results in increased T cell receptor-mediated activation of the Rho/Rac exchange factor Vav and of the JNK-p38 MAPK signaling pathway. The effects of Cybr on nuclear factor of activated T cells and AP-1 are dependent on MAPK activation, and enhanced activation of this cascade results in cooperation between the two transcription factors in the regulation of gene expression. These findings provide the first evidence that the adaptor protein Cybr not only regulates lymphocyte adhesion and cell-cell interaction but also contributes to the regulation of the signaling cascade and of the genetic program downstream of the T cell receptor.

Characterization of the varicella zoster virus (VZV)-specific intra-ocular T-cell response in patients with VZV-induced uveitis

Varicella zoster virus (VZV) is a well-known cause of infectious uveitis. The aim of this study was to characterize the VZV-specific T-cell response in eyes of patients with VZV-induced uveitis. T-cell lines (TCL) were generated by mitogenic stimulation of intra-ocular fluid (IOF) samples obtained from eight patients with VZV-induced uveitis. Two patients with herpes simplex virus (HSV)-induced uveitis were included as disease controls. Characterization of individual T-cells in the TCL was performed by stimulating the TCL with mock, HSV-1 and VZV antigen pulsed autologous B cells and subsequent flow cytometric analyses. Virus specificity and phenotype of the T-cells were identified by simultaneous detection of intracellular gamma interferon and cell surface markers CD4, CD8, CD3 or T-cell receptor (TCR) beta chain variable region (TCRBV) usage. The TCL obtained from patients with HSV-1-induced uveitis contained higher numbers of T-cells reactive to HSV-1 compared to VZV. VZV-specific T-cells were detected in all TCL of the patients diagnosed with VZV-induced uveitis. Four out of six TCL obtained from patients with VZV-induced uveitis that were assayed for both viruses, contained higher numbers of T-cells reactive to VZV compared to HSV-1. Detailed analyses of the TCL of two patients demonstrated that the VZV reactivity within the assayed TCL was dominated by T-cells expressing one specific TCRBV gene. The data implicate that VZV-reactive T-cells infiltrate and participate in the local inflammatory response in eyes of patients with VZV-induced uveitis.

Quantitative Analysis of Phosphotyrosine Signaling Networks Triggered by CD3 and CD28 Costimulation in Jurkat Cells

The mechanism by which stimulation of coreceptors such as CD28 contributes to full activation of TCR signaling pathways has been intensively studied, yet quantitative measurement of costimulation effects on functional TCR signaling networks has been lacking. In this study, phosphotyrosine networks triggered by CD3, CD28, or CD3 and CD28 costimulation were analyzed by site-specific quantitative phosphoproteomics, resulting in identification of 101 tyrosine and 3 threonine phosphorylation sites and quantification of 87 sites across four cell states. As expected, CD3 stimulation induced phosphorylation of CD3 chains and upstream components of TCR pathways such as Zap70, while CD28 stimulation induced phosphorylation of CD28, Vav-1, and other adaptor proteins including downstream of tyrosine kinase 1, Grb2-associated protein 2 (Grap2), and Wiskott-Aldrich syndrome protein. CD3 and CD28 costimulation induced a complex response including decreased threonine phosphorylation in the ERK1 and ERK2 activation loops and increased phosphorylation of selected tyrosine sites on ERK1/2, p38, phospholipase C-gamma, Src homology 2 domain-containing transforming protein 1, Grap2, and Vav-1, potentiating T cell activation. Hierarchical clustering and self-organizing maps were used to identify modules of coregulated phosphorylation sites within the network. Quantitative information in our study suggests quantitative and qualitative contribution by costimulation of CD28 on CD3-stimulated TCR signaling networks via enhanced phosphorylation of phospholipase C-gamma/Src homology 2 domain-containing transforming protein 1/Grap2/Vav-1 and their effects on downstream components including MAPKs.

Stoichiometry of the murine gammadelta T cell receptor.

The T cell receptor for antigen (TCR) complex is organized into two functional domains: the antigen-binding clonotypic heterodimer and the signal-transducing invariant CD3 and TCRζ chains. In most vertebrates, there are two different clonotypic heterodimers (TCRαβ and TCRγδ) that define the αβ and γδ T cell lineages, respectively. αβ- and γδTCRs also differ in their invariant chain subunit composition, in that αβTCRs contain CD3γɛ and CD3δɛ dimers, whereas γδTCRs contain only CD3γɛ dimers. This difference in subunit composition of the αβ- and γδTCRs raises the question of whether the stoichiometries of these receptor complexes are different. As the stoichiometry of the murine γδTCR has not been previously investigated, we used two quantitative immunofluorescent approaches to determine the valency of TCRγδ heterodimers and CD3γɛ dimers in surface murine γδTCR complexes. Our results support a model of murine γδTCR stoichiometry in which there are two CD3γɛ dimers for every TCRγδ heterodimer.

Critical Function for SIP, a Ubiquitin E3 Ligase Component of the β-Catenin Degradation Pathway, for Thymocyte Development and G1 Checkpoint

Beta-catenin has been implicated in thymocyte development because of its function as a coactivator of Tcf/LEF-family transcription factors. Previously, we discovered a novel pathway for p53-induced beta-catenin degradation through a ubiquitin E3 ligase complex involving Siah1, SIP (CacyBP), Skp1, and Ebi. To gain insights into the physiological relevance of this new degradation pathway in vivo, we generated mutant mice lacking SIP. We demonstrate here that SIP-/- thymocytes have an impaired pre-TCR checkpoint with failure of TCRbeta gene rearrangement and increased apoptosis, resulting in reduced cellularity of the thymus. Moreover, the degradation of beta-catenin in response to DNA damage is significantly impaired in SIP-/- cells. SIP-/- embryonic fibroblasts show a growth-rate increase resulting from defects in G1 arrest. Thus, the beta-catenin degradation pathway mediated by SIP defines an essential checkpoint for thymocyte development and cell-cycle progression.

Development of Retargeted CD38-Specific NK-92 Cell Line for Potential Anti-Myeloma Immunotherapy.

Multiple Myeloma (MM) is a B cell neoplasm in which malignant plasma cells accumulate in the bone marrow and produce excessive amounts of a monoclonal immunoglobulin. Among the large number of antigens expressed by plasma cells, CD38 is one of the highest expressed surface molecules, suggesting an alternative public tumour target. For instance, the myeloma cell line 8226 has a high surface expression of CD38. We plan to introduce genetically recombinant single-chain variable fragments (scFv) of anti-CD38 antibody, along with CD3 chain, into NK-92 a natural killer cell line which is highly cytotoxic against a variety of malignant cells (the only natural killer cell line being used in phase I/II clinical trials). It is likely that the retargeted NK-92 cells may specifically and efficiently kill CD38-expressing myeloma cells. By using a series of degenerate primer sets for mouse immunoglobulin variable region genes, VH and VL regions were isolated through reverse transcription PCR from OKT10 cell line. Further sequencing analysis provided novel information of the OKT10 immunoglobulin heavy and light chains. A PCR-based assembling strategy was employed to construct the scFv with FLAG sequence integrated as a tag. VH and VL domains were joined together with a hinge region of (Gly4Ser)3. The phagemid construct has been formed by subcloning the scFv fragment into the Sfi I/Not I cloning sites of the expression vector pHEN2, and subsequently transformed into the E. Coli HB2151. Five out of 7 transformed bacterial colonies screened did not contain the scFv fragment. For the remaining two clones with the correct-sized insert, we tested the specificity of the scFv fragments that were prepared through intracellular antibody capture as periplasmic proteins. We performed a competitive flow assay using periplasmic proteins with commercially available anti-CD38 PE conjugated antibodies on 8226 myeloma cell line. We found that one of these two clones (clone 6) showed a reduction in CD38 staining and presented as double peaks; while this pattern was not seen in the clone 7. Subsequent DNA sequencing revealed that there was a 100-bp truncation in the heavy chain region in clone 7; while the clone 6 has a complete productive heavy chain and light chain spanned by the linker region. We are going to transfect NK-92 cells with retrovial vector pL-scFv(clone 6) construct as the basis of an efficient approach for anti-myelome immunotherapy. (Dr. Daniel Sze is supported by the International Myeloma Foundation Senior Research Grant).

Extramedullary Plasmacytoma of the Salivary Gland in Two Syrian Hamsters (Mesocricetus auratus)

Two Syrian hamsters developed marked swelling of the ventral neck. Histologic examination of both masses revealed that the submaxillary salivary glands were effaced by large numbers of neoplastic plasma cells. In one hamster, neoplastic cells had infiltrated the adjacent lymph node. The neoplastic cells expressed CD79a antigen and were negative for CD3, lambda, and kappa light chains. Ultrastructural features of neoplastic cells in the salivary gland of one hamster included abundant cytoplasmic rough endoplasmic reticulum profiles, and peripherally displaced nuclei that contained marginated heterochromatin, consistent with plasma cells. Salivary gland plasmacytomas are extremely rare in humans and have not previously been reported in nonhuman species. The occurrence of such neoplasms in two hamsters suggests that this species may be predisposed to developing tumors of this type.