CD3epsilon Research Articles

Advances in the management and monitoring of extranodal NK/T‐cell lymphoma, nasal type

Extranodal natural killer (NK)/T-cell lymphoma, nasal type, has a unique geographic distribution. Its pathology is characterized by marked angio-invasion and tissue necrosis. A typical NK-cell phenotype is usually present: CD2(+), CD3 epsilon+, CD56(+), cytotoxic molecules+ and Epstein-Barr virus (EBV)+. Magnetic Resonance Imaging helps to clearly define the local involvement. Positron Emission Tomography helps to demonstrate system spread. Various prognostic variables (International Prognostic Index or the Korean Prognostic Index) should be documented. This may include quantification of plasma EBV DNA. For localized nasal disease, radiotherapy is important, although chemotherapy is often added. Sustainable remission is observed in over half of these patients. For extra-nasal or disseminated disease, systemic chemotherapy becomes the mainstay and the prognosis is usually poor. Doxorubicin-containing regimens are not entirely satisfactory and L-asparaginase containing regimens are being investigated. Patients with poor prognostic features may be considered for an early autologous haematopoietic stem cell transplant. Allogeneic transplantation is efficacious but is associated with high transplant-related mortality.

In vitro preparation and characterization of the human CD3εε homodimer and CD3εγ and CD3εδ heterodimers

The CD3 molecule is a critical component of both humoral and cellular immune responses, and yet while the structure and molecular assembly of other key mediators such as CD4 and CD8 have been reported, individual CD3 subunits have not been well characterized. Our understanding of the manner in which they interact remains limited, and the question of how many subunits are required for a functional CD3 molecular complex is yet to be addressed. It has been suggested that CD3 epsilon pairs with CD3 gamma or with CD3 delta, forming CD3 epsilon gamma and CD3 epsilon delta heterodimers that associate with alpha/beta T cell receptors (TCRs) and CD3 zeta 2 dimers. In this study we investigated whether interactions between each CD3 epsilon subunit play a role in the formation of the CD3 molecular complex. Our results revealed that the human CD3 epsilon subunit forms a homodimer structure, which is a crucial piece of information for the elucidation of cellular signaling following TCR receptor ligation, and provide insight into our understanding of the molecular assembly of the CD3 molecular complex.

Neurobehavioral Performance in Feline Immunodeficiency Virus Infection: Integrated Analysis of Viral Burden, Neuroinflammation, and Neuronal Injury in Cortex

Human immunodeficiency virus (HIV) infection causes motor and neurocognitive abnormalities affecting >50% of children and 20% of adults with HIV/AIDS (acquired immunodeficiency syndrome). The closely related lentivirus, feline immunodeficiency virus (FIV), also causes neurobehavioral deficits. Herein, we investigated the extent to which FIV infection affected specific motor and cognitive tasks in conjunction with viral burden and immune responses within the brain. Neonatal animals were infected with a neurovirulent FIV strain (FIV-Ch) and assessed in terms of systemic immune parameters, viral burden, neurobehavioral performance, and neuropathological features. FIV-infected animals displayed less weight gain and lower blood CD4(+) T-cell levels than mock-infected animals (p < 0.05). Gait analyses disclosed greater gait width with increased variation in FIV-infected animals (p < 0.05). Maze performance showed that FIV-infected animals were slower and made more navigational errors than mock-infected animals (p < 0.05). In the object memory test, the FIV-infected group exhibited fewer successful steps with more trajectory errors compared with the mock-infected group (p < 0.05). Performance on the gait, maze, and object memory tests was inversely correlated with F4/80 and CD3 epsilon expression (p < 0.05) and with viral burden in parietal cortex (p < 0.05). Amino acid analysis in cortex showed that D-serine levels were reduced in FIV-infected animals, which was accompanied by diminished kainate and AMPA receptor subunit expression (p < 0.05). The neurobehavioral findings in FIV-infected animals were associated with increased gliosis and reduced cortical neuronal counts (p < 0.05). The present studies indicated that specific motor and neurocognitive abilities were impaired in FIV infection and that these effects were closely coupled with viral burden, neuroinflammation, and neuronal loss.

The cytoplasmic tail of the T cell receptor CD3 epsilon subunit contains a phospholipid-binding motif that regulates T cell functions.

The CD3 epsilon subunit of the TCR complex contains two defined signaling domains, a proline-rich sequence and an ITAM. We identified a third signaling sequence in CD3 epsilon, termed the basic-rich stretch (BRS). Herein, we show that the positively charged residues of the BRS enable this region of CD3 epsilon to complex a subset of acidic phospholipids, including PI(3)P, PI(4)P, PI(5)P, PI(3,4,5)P(3), and PI(4,5)P(2). Transgenic mice containing mutations of the BRS exhibited varying developmental defects, ranging from reduced thymic cellularity to a complete block in T cell development. Peripheral T cells from BRS-modified mice also exhibited several defects, including decreased TCR surface expression, reduced TCR-mediated signaling responses to agonist peptide-loaded APCs, and delayed CD3 epsilon localization to the immunological synapse. Overall, these findings demonstrate a functional role for the CD3 epsilon lipid-binding domain in T cell biology.

Nasal natural killer (NK)/T-cell lymphoma: clinical, histological, virological, and genetic features

Nasal natural killer (NK)/T-cell lymphoma (NNKTL) is a clinical illness characterized by progressive unrelenting ulceration and necrosis of the nasal cavity and midline facial tissues. Histological features of the lymphoma include angiocentric and polymorphous lymphoreticular infiltrates, called polymorphic reticulosis. Surface antigens and the NK-cell marker, CD56, as well as pan-T antigen CD2, cytoplasmic CD3 (CD3epsilon), and CD45 are expressed in the lymphoma cells. The origin of the lymphoma is thought to be either NK-cell linkage without T-cell receptor (TCR) rearrangement or gammadeltaT-cell linkage with gammadeltaTCR rearrangement. Since the authors of this study first demonstrated the presence of Epstein Barr virus (EBV)-DNA and EBV oncogenic proteins in NNKTL, the lymphoma has been classified as one of the EBV-associated malignancies. The NNKTL cells produce interleukin (IL)-9, IL-10, and interferon-gamma-inducible protein-10 (IP-10), possibly due to EBV-oncogenic proteins in the lymphoma cells, and such cytokines take an important part in the cell proliferation and invasion, acting in an autocrine manner. Clinically, the serum EBV-DNA copy number is useful as a specific tumor marker and a predictive prognostic factor. Even in early clinical stages, the lymphoma shows poor prognosis caused by the rapid progression of the lesion into distinct organs. Our newly designed arterial infusion chemotherapy, from the superficial temporal artery, in combination with radiotherapy, has shown a favorable outcome in patients with NNKTL. In this article, the clinical, pathological, and virological characteristics of the lymphoma are reviewed, along with a report of our investigations.

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.

Susceptibility to type I diabetes in women is associated with the CD3 epsilon locus on chromosome 11

Type I diabetes is associated with the DQ loci of the MHC and to a lesser extent with the T cell antigen receptor (TcR) beta chain genes. The non-obese diabetic (NOD) mouse is an animal model of human diabetes, in which up to 90% of female mice develop overt insulin-dependent diabetes. Genetic studies in the NOD mouse suggest that there are at least three diabetogenic genes; one that maps to the MHC, another that may map to the mouse Thy-I locus, and a third that has still to be identified. We have investigated loci in the vicinity of the human Thy-I locus on chromosome 11q23 and report here the results of restriction fragment length polymorphism (RFLP) analysis of the CD3 epsilon locus of 168 Caucasoid patients with type I diabetes. While no association was found between this locus and type I diabetes, a significant difference in the frequency of the CD3 epsilon 8-kb allele was found between male and female patients (0.268 versus 0.430; P less than 0.0025, Pc = 0.02) and between female patients and healthy female controls (0.430 versus 0.267; P less than 0.015). These results suggest that a gene residing on chromosome 11q23 may confer susceptibility to type I diabetes in women.

Intravascular Large T-cell or NK-cell Lymphoma

Most cases of intravascular large cell lymphoma have a B-cell phenotype, but rare T-cell and natural killer (NK)-cell variants have been reported. We describe the clinicopathologic features of 4 patients (M:F=3:1; age range: 63 to 87; median age: 65) with intravascular large NK/T-cell lymphoma. The skin was the site of presentation in all patients (leg: 1 case; trunk: 1 case; trunk and extremities: 2 cases). Two patients had lesions confined to the skin; in 1 case concomitant involvement of the brain was detected and in 1 case no further studies were carried out. Immunohistology showed positivity for cytotoxic markers in 3/4 cases. One case had an NK phenotype similar to NK/T-cell lymphoma, nasal-type, whereas the other cases could not be precisely classified into specific categories (peripheral T-cell lymphoma, NOS). One of these cases was negative for cytotoxic markers and was positive only for CD2 and CD3 epsilon. Association with Epstein-Barr virus (EBV) was demonstrated in 2 cases by in situ hybridization, whereas 1 case was negative. All our patients had aggressive disease and died between 2 weeks and 7 months from presentation. Analysis of our cases and of those published in the literature shows that intravascular large NK/T-cell lymphoma is a rare, aggressive lymphoma with variable phenotypic features, frequent expression of cytotoxic proteins, true NK-cell phenotype and association with Epstein-Barr virus infection, and common presentation in the skin. Homogeneous studies on larger number of patients and reevaluation of cases published with incomplete phenotypic data would be necessary to gather more information on this extremely rare type of lymphoma.

Flow cytometric analysis of intestinal intraepithelial lymphocytes in the diagnosis of refractory celiac sprue

The etiology of refractory celiac sprue (RCS) is unclear. In a high proportion of cases, the clonal nature of intestinal intraepithelial lymphocytes (IEL) can be demonstrated and a pathogenetic implication of intestinal IEL has been postulated. The prognosis of this subgroup of RCS is poor, with a high risk to develop an overt lymphoma and uncontrolled malabsorption despite steroid/immunosuppressive therapy. Cases with a relatively indolent clinical course, however, exist and their early diagnosis may be difficult. To gain insight into the pathogenic implication of intestinal IEL in refractory celiac sprue, we have performed an extensive phenotypic and functional characterization of clonal intestinal IEL in a patient with an indolent form of refractory celiac sprue, using multiparametric flow cytometry. The abnormal lymphocyte infiltrate lacked surface membrane expression of CD3/T-cell receptor (TCR) complexes (TCR(-), CD4(-), CD8(-), sCD3(-)), but contained intracellular CD3(epsilon) (CyCD3(+)) and surface CD103(+) and CD7(+). In particular, these cells showed a unique spontaneous ex-vivo cytokine secretion profile with an increased percentage of CD3(-) IEL containing TNF-alpha and IL-10, in the absence of IL-2, IL-4 and IFN-gamma. Altogether our results suggest that flow cytometry immunophenotyping of intestinal IEL, in cases suspected of celiac disease and their complicated forms, could be of great help in the correct diagnosis of RCS and the understanding of the immunopathogenic mechanisms of the disease and their clinical and/or therapeutical implications.

Antibody microarray analysis of cell surface antigens on CD4+ and CD8+ T cells from HIV+ individuals correlates with disease stages

BackgroundExpression levels of cell surface antigens such as CD38 and HLA-DR are related to HIV disease stages. To date, the immunophenotyping of cell surface antigens relies on flow cytometry, allowing estimation of 3–6 markers at a time. The recently described DotScan antibody microarray technology enables the simultaneous analysis of a large number of cell surface antigens. This new technology provides new opportunities to identify novel differential markers expressed or co-expressed on CD4+ and CD8+ T cells, which could aid in defining the stage of evolution of HIV infection and the immune status of the patient.ResultsUsing this new technology, we compared cell surface antigen expression on purified CD4+ and CD8+ T cells between 3 HIV disease groups (long-term non-progressors controlling viremia naturally; HIV+ patients on highly active antiretroviral therapy (HAART) with HIV plasma viral loads <50 copies/ml; and HIV+ patients with viremia during HAART) and uninfected controls. Pairwise comparisons identified 17 statistically differential cell surface antigens including 5 novel ones (CD212b1, CD218a, CD183, CD3 epsilon and CD9), not previously reported. Notably, changes in activation marker expression were more pronounced in CD8+ T cells, whereas changes in the expression of cell membrane receptors for cytokines and chemokines were more pronounced in CD4+ T cells.ConclusionOur study not only confirmed cell surface antigens previously reported to be related to HIV disease stages, but also identified 5 novel ones. Of these five, three markers point to major changes in responsiveness to certain cytokines, which are involved in Th1 responses. For the first time our study shows how density of cell surface antigens could be efficiently exploited in an array manner in relation to HIV disease stages. This new platform of identifying disease markers can be further extended to study other diseases.

The Sooner the Better: Rapid Transduction Enhances the Engraftment/Selection of Gene Corrected Fanconi Anemia HSC.

Fanconi anemia (FA) is amenable to genetic correction of hematopoietic stem cells (HSCs). However, as demonstrated in previous clinical gene therapy trials, successful extension of murine studies into human therapies is limited by low numbers of target HSC and poor engraftment of transduced FA HSC (Kelly et al., Mol Ther, 2007). To examine the potential biological consequences/benefits of shortened transduction we used a FA mouse model in which HSC are deficient and prone to excessive loss during in vitro manipulation. We applied a rapid transduction protocol (Mostoslavsky et al., Mol Ther, 2005) utilizing lentiviral vectors and demonstrate that this shortened transduction preserves engraftment of FA HSC to the level of C57BL/6 wt cells. Lin− Sca-1+ c-Kit+ bone marrow cells were isolated from Fanca−/− CD45.2 mice and underwent 4-hr rapid (RT) vs. 96-hr conventional (CT) transduction. An equivalent number of transduced cells were transplanted into lethally irradiated CD45.1 BoyJ mice. Analysis of engraftment chimerism three months post transplantation revealed a significantly higher level of engraftment in animals receiving RT vs. CT cells (90% +/− 14% vs. 26% +/− 31%, respectively, p=<0.01). Rapid transduction also resulted in a significant reduction of engraftment failure (0/36 animals RT vs. 20/36 animals CT). Importantly--emphasizing the FA disease-specific stem cell phenotype, RT vs. CT of C57BL/6 wt cells was associated with no significant difference in engraftment of these cells (93% +/− 1.2% RT vs. 84 +/− 19% CT, p=0.33). Analysis of peripheral blood cells expressing the proviral enhanced green fluorescent protein (eGFP) reporter gene revealed a normal distribution of B-lymphocytes (B220), T-lymphocytes (CD3 epsilon), and granulocytes (MAC-1), indicating multi-lineage engraftment of gene modified cells. In spite of this engraftment advantage, transduction efficiency was low (<30%) using RT. The 6-benzylguanine (6-BG) resistant P140K mutant of O6-methylguanine DNA methyltransferase (MGMTP140K) confers a selective advantage to tranduced HSC treated with alkylating drugs. Following RT with a MGMTP140K/ eGFP expressing lentivirus, 5/6 mice treated with 6-BG and the alkylating drug temozolomide showed a significant rise in the percentage of GFP reporter gene expression in peripheral blood. We extended this approach to the FA model by generating a tri-cistronic lentiviral vector expressing the FANCA cDNA, MGMTP140K, and eGFP. Despite modest in vivo gene marking with this vector, up to 37-fold selection (85% GFP-positive cells) was achieved following exposure of bone marrow of transplant recipients to 6-BG and the alkylating drug temozolomide in vitro. Concurrently, phenotypic correction of mitomycin C hypersensitivity of transduced Fanca−/− bone marrow cells was observed. These data suggest that RT improves stem cell engrafting capacity of FA stem cells in a relevant animal model of stem cell gene therapy. The combination of RT and in vivo selection may allow more successful reconstitution of the lympho-hematopoietic system in gene therapy applications.

Lung carcinomas do not induce T-cell apoptosis via the Fas/Fas ligand pathway but down-regulate CD3 epsilon expression.

Non-small cell lung carcinoma (NSCLC) patients have impaired cellular immune responses. It has been hypothesized that tumor cells expressing Fas Ligand (FasL) induce in T lymphocytes: (a) apoptosis (tumor counterattack) and (b) down-regulation of CD3zeta expression. However, the hypothesis of tumor counterattack is still controversial. We analyzed FasL expression on NSCLC cell lines and on tumor cells from lung adenocarcinoma patients by flow cytometry and immunocytochemistry. FasL mRNA expression was detected in NSCLC cell lines using RT-PCR, and functional FasL was evaluated on Fas-expressing Jurkat T-cells by annexin-V-FITC staining and by SubG(1) peak detection. Also, the proapoptotic effect of microvesicles released from NSCLC cell lines in Jurkat T-cells was studied. Alterations in the expression levels of CD3zeta, CD3epsilon, and CD28 [measured as mean fluorescence intensity (MFI)] were determined in Jurkat T-cells after co-culture with NSCLC cell lines or tumor-derived microvesicles. Furthermore, the expression levels of CD3zeta and CD3epsilon in CD4+T and CD8+T lymphocytes from lung adenocarcinoma patients was studied. Our results indicate that NSCLC cells neither FasL expressed nor induced apoptosis in Jurkat T-cells. Tumor-derived microvesicles did not induce apoptosis in Jurkat T-cells. In contrast, NSCLC cell lines down-regulated CD3epsilon but not CD3zeta chain expression in Jurkat T-cells; this effect was induced by soluble factors but not by microvesicles. In lung adenocarcinoma patients, significant decreases of MFI values for CD3epsilon, but not CD3zeta, were found in CD4+T and CD8+T cells from pleural effusion compared to peripheral blood and in peripheral blood of patients compared to healthy donors. Our data do not support the tumor counterattack hypothesis for NSCLC. Nonetheless, down-regulation of CD3epsilon in T-cells induced by NSCLC cells might lead to T-cell dysfunction.

Allosteric Changes in the TCR/CD3 Structure Upon Interaction With Extra‐ or Intra‐cellular Ligands

T lymphocytes are activated by the interaction between the T-cell antigen receptor (TCR) and peptides presented by major histocompatibility complex (MHC) molecules. The avidity of this TCR-pMHC interaction is very low. Therefore, several hypotheses have been put forward to explain how T cells become specifically activated despite this handicap: conformational change model, aggregation model, kinetic segregation model, sequential interaction model and permissive geometry model. In the present paper, we conducted experiments to distinguish between the TCR-aggregation model and the TCR-conformational change model. The results obtained using a TCR capture ELISA with Brij 98-solubilized TCR molecules from normal or activated T cells showed that the ligand-TCR interaction causes structural changes in the CD3 epsilon cytoplasmic tail as well as in the extracellular TCR beta FG loop region. Size-fractionation experiments with Brij 98-solubilized TCR/CD3/co-receptor complexes from naïve or activated CD4(+) or CD8(+) T cells demonstrated that such complexes are found as either dimers or tetramers. No monomers or multimers were detected. We propose that: (1) ligand-TCR interaction results in conformational changes in the CD3 epsilon cytoplasmic tail leading to T-cell activation; (2) CD3 epsilon cytoplasmic tail interaction with intracellular proteins may dissociate pMHC and co-receptors (CD4 or CD8) from TCR/CD3 complexes, thus leading to the arrest of T-cell activation; and (3) T-cell activation appears to occur among dimers or tetramers of TCR/CD3/co-receptor complexes interacting with self and non-self (foreign) peptide-MHC complexes.

The Membrane-proximal Portion of CD3 ε Associates with the Serine/Threonine Kinase GRK2

The activation of protein kinases is one of the primary mechanisms whereby T cell receptors (TCR) propagate intracellular signals. To date, the majority of kinases known to be involved in the early stages of TCR signaling are protein-tyrosine kinases such as Lck, Fyn, and ZAP-70. Here we report a constitutive association between the TCR and a serine/threonine kinase, which was mediated through the membrane-proximal portion of CD3 epsilon. Mass spectrometry analysis of CD3 epsilon-associated proteins identified G protein-coupled receptor kinase 2 (GRK2) as a candidate Ser/Thr kinase. Transient transfection assays and Western blot analysis verified the ability of GRK2 to interact with the cytoplasmic domain of CD3 epsilon within a cell. These findings are consistent with recent reports demonstrating the ability of certain G protein-coupled receptors (GPCR) and G proteins to physically associate with the alpha/beta TCR. Because GRK2 is primarily involved in arresting GPCR signals, its interaction with CD3 epsilon may provide a novel means whereby the TCR can negatively regulate signals generated through GPCRs.

LAT Down-Regulation in T Lymphocytes from B-Cell Chronic Lymphocytic Leukemia: A Possible Mechanism for T Cell Incompetence.

Patients with chronic lymphocytic leukaemia (CLL) show, in addition to the clonal expansion of the malignant B cells, several abnormalities in the non-malignant T cells population. These include T cell lymphocytosis, an increase of CD8+ T cells which results in a low CD4/CD8 ratio and a defective proliferative response to mitogenic stimuli.A down-regulation of TCR-related CD3 zeta protein in T cells of CLL has been reported suggesting that T cell immune deficiency may result from an impairment of cell activation machinery. In addition recent data show a decreased level of the costimolatory CD28 molecules and a concomitant augment of the downregulatory CTLA-4 molecules suggesting that T cells may be in a partial state of anergy which may account for their failure to display a full activation response following TCR engagement.Despite the evidence of a compromission in theT cell signal machinery, little is known about possible defects in other molecules involved in the cell signal transduction pathway.In order to investigate the expression of the proteins involved in the early TCR signalling events CD3+ T cells of 7 healthy donors and of 21 B-CLL patients were purified by negative selection using magnetic beads (purity >95%) and lysed in 1% Triton X-100. Subsequently, equal amounts of protein for each sample were analyzed by Western Blotting and the following molecules were evaluated: CD3 epsilon, p56Lck (Lck) and linker for activation of T cells (LAT).Overall results showed that CD3 epsilon in T cells of B-CLL was expressed in an amount comparable to normal healthy donors in 15/21 (71%) cases while was reduced in 6/21 (29%), Lck was reduced in 3/21 (14%) cases and interestingly LAT was reduced in 15/21 (71%) cases. Our data show that, apart from CD3 zeta, other molecules involved in the early activation events, are reduced in T cells of B-CLL. CD3 epsilon was reduced in a third of the cases tested. This subunit, along with CD3 zeta, plays a crucial role in the early TCR signalling events, its reduction therefore may contribute to explain the hyporesponsiveness showd by T lymphocytes.Interestingly we observed a significant reduction in the expression of LAT molecule. LAT is an adaptor molecule which, even if lacking in enzymatic activity partecipates to cell signalling acting as a plasma membrane scaffold. This molecule following TCR engagement is phosphorilated by ZAP-70 kinase and in the active form promotes the recruitment at the plasma membrane of molecules with SH2 domains and the assembly of numerous signalling complexes. LAT reduced expression in T cell is of particular interest in the light of recent data, obtained in mice, showing that anergic T cells are defective in LAT activation.To our best knowledge so far there are no available biochemical data about abnormalities in the expression degree of trasduction molecules in human anergic T cells, so our data may represent a preliminary contribution in understanding a possible molecular mechanism at the basis of the anergic condition displayed by T cell compartment of B-CLL.

Immunological markers of outcome of non-small cell lung cancer

7232 Background Previously, we identified by cDNA microarray analysis that upregulation of immune synapse markers, including CD2, CD3 epsilon, T cell receptor, CD28, HLA class I and II and CD86 cor...

CD3δ immunodeficiency

The review describes advances in understanding the role of the CD3 delta subunit in human T-cell development as deduced from a recently described human immunodeficiency. The review also compares CD3 delta deficiency with other human CD3 subunit deficiencies and with corresponding animal models. In describing CD3 delta deficiency in humans this review shows that patients with profound T-cell depletion, who present at 2-3 months with severe viral infection, lack CD3 delta as a result of a mutation in the extracellular domain of this gene. The genetic aberration was discovered by comparing patients' and normal thymocytes, using mass gene screening with the microarray technique. In humans the absence of CD3 delta results in a complete arrest in thymocyte development at the stage of double negative to double positive transition and the development of gamma delta T-cell receptor-positive T cells is also impaired. Unlike patients with CD3 gamma or CD3 epsilon deficiency who have a milder condition, patients with CD3 delta deficiency present with severe lethal susceptibility to infections during early infancy. As expected, this profound immunodeficiency was cured with an allogenic bone marrow transplantation. In contrast to murine CD3 (-/) delta, which retains a normal gamma delta T-cell receptor-positive T-cell population and only partly affects the developmental transition of double positive to single positive thymocytes, CD3 delta in humans appears to be more critically required for the development of both alpha beta and gamma delta T-cell receptor-positive T-cell lineages. The studies also show for the first time that comparing relevant patients' with normal tissue using microarray technology can aid in the discovery of the genetic basis of inherited disorders.

Molecular modification of a recombinant, bivalent anti-human CD3 immunotoxin (Bic3) results in reduced in vivo toxicity in mice

A novel bivalent single chain fusion protein, Bic3, was assembled consisting of the catalytic and translocation domains of diphtheria toxin (DT 390) fused to two repeating sFv molecules recognizing human CD3 epsilon of the human T-cell receptor. Historically, problems with these constructs include low yield, toxicity, and reduced efficacy. Instead of using conventional Gly 4Ser linkers to connect heavy/light chains, aggregation reducing linkers (ARL) were used which when combined with a new SLS-based refolding method reduced aggregation and enhanced the yield of final product. Toxicity was reduced at least 25-fold by repeating the two sFv molecules and adding a portion of the hinge-CH2–CH3 human constant regions. The resulting Bic3 was just as cytotoxic to HPB-MLT.UM T leukemia cells in vitro (IC 50 = 4 pmol) as a monovalent construct made with the same DT and sFv. In vivo, Bic3 was effective in a new and aggressive therapy model in which it significantly prolonged survival of scid mice with established human T-cell leukemia ( p < 0.0001 compared to controls). Importantly, no toxicity measured by weight loss, enzyme function, or histology was observed at the highest dose of Bic3 tested (2000 ug/kg). Bic3 warrants investigation as a new drug for treating T-cell malignancy and other T-cell related disorders.

Efficient induction of T helper type 1-mediated immune responses in antigen-primed mice by anti-CD3 single-chain Fv/interleukin-18 fusion DNA.

Two types of T helper (Th) cells - Th1 and Th2 - play different roles in protection and immunopathology. The Th1 cell-mediated immune response plays an important role in inducing the host defence against intracellular bacteria and also in cancer immunotherapy. To effectively induce Th1 immune responses, we constructed a mammalian expression plasmid (pAnti-CD3sFv/IL-18) carrying a fusion gene in which anti-CD3 single-chain Fv (sFv) cDNA, the smallest unit of antibody recognizing the CD3 epsilon moiety of the T-cell receptor, was covalently linked to mature interleukin (IL)-18 cDNA. Intramuscular injection of ovalbumin (OVA)-sensitized BALB/c mice with pAnti-CD3sFv/IL-18 DNA efficiently increased the production of both OVA-specific interferon-gamma and anti-OVA immunoglobulin G2a, compared to injection with pAnti-CD3sFv DNA. In addition, pAnti-CD3sFv/IL-18 was more efficient than a mixture of pAnti-CD3sFv + pIL-18 in inducing OVA-specific, Th1 immune responses and also in inhibiting OVA-specific, IL-4 production. These studies indicate that vaccination with pAnti-CD3sFv/IL-18 fusion DNA efficiently induces the Th1 immune response in antigen-sensitized mice.

Induction of human T lymphocyte cytotoxicity and inhibition of tumor growth by tumor-specific diabody-based molecules secreted from gene-modified bystander cells.

Infiltrating T cells are found in many malignancies, but they appear to be mostly anergic and do not attack the tumor, presumably because of the absence of activation and/or costimulatory signals. We describe a strategy for cellular antitumor immunotherapy by the in situ production of soluble bifunctional Ab-based molecules that activate and retarget T cells to the tumor. We genetically modified cells to simultaneously secrete two bifunctional molecules, a bispecific diabody directed against the carcinoembryonic Ag (CEA) and the CD3 epsilon chain of the TCR (alphaCEA x alphaCD3), and a fusion protein comprising the extracellular portion of B7-1 fused to a bivalent anti-CEA diabody (B7-alphaCEA). Together, alphaCEA x alphaCD3 and B7-alphaCEA proved potent at inducing the activation, proliferation, and survival of primary human T cells. When producer cells were cocultured with primary T cells and CEA(+) cancer cells, alphaCEA x alphaCD3 and B7-alphaCEA acted in combination to activate and retarget T cell cytotoxicity and completely abrogate tumor growth in the coculture. Furthermore, the introduction of just a few such producer cells at the tumor site efficiently inhibited the growth of established human colon carcinoma xenografts. Despite a cumbersome generation process, the use of autologous gene-modified producer cells opens the way for a new diabody-based gene therapy strategy of cancer.