CD3 Zeta Chain Research Articles

CD3 zeta chain deficient T cells instigate multi-organ tissue inflammation (123.35)

Abstract A population of T cells that lack the CD3 zeta (ζ) chain exists in systemic lupus erythematosus (SLE), rheumatoid arthritis, cancer, and infectious diseases. SLE is a chronic autoimmune disease characterized by high production of autoantibodies and multi-organ tissue inflammation. However, the role of CD3ζ chain-deficient T cells in the pathogenesis of these disorders remains unknown. Here we show the role of CD3ζ chain deficient T cells in multi-organ tissue inflammation. We found that CD3ζ -deficient mice spontaneously developed multi-organ tissue inflammation that can be promoted following the injection of poly: (ic) or allogeneic cells. Interestingly, a humoral response did not accompany the cellular response. CD3ζ chain deficient T cells were found defective in activation-induced apoptosis, and to display aggregated lipid rafts in which CD44, and CCR2 colocalized and to produce IFN-γ. Anti-IFN-γ treatment of CD3ζ deficient mice inhibited spontaneous multi-organ tissue inflammation. Promotion and resolution of lipid raft aggregation resulted in enhanced and decreased IFN-γ production suggesting a clear role of aggregated lipid rafts in the production of IFN-γ. Our results demonstrate that CD3ζ chain deficient T cells instigate multi-organ tissue inflammation by enabling lipid raft aggregation. Thus we demonstrate that decreased CD3ζ chain expression in autoimmunity, cancer and infection, contributes independently to inflammation and damage.

The Splicing Factor ASF/SF2 enhances Interleukin-2 production in human T lymphocytes (121.5)

Abstract Systemic lupus erythematosus (SLE) is a complex autoimmune disease which afflicts mainly women in the reproductive years, and causes painful arthritis, skin disease and complications in the kidneys and brain. Abnormal T lymphocytes in SLE not only regulate autoantibody-producing B cells, but are also responsible for target organ infiltration. Currently, no specific therapeutic strategies target T cell defects. Moreover, there are no known molecular markers to predict disease activity. Aberrant molecular mechanisms responsible for SLE T cell defects are thus promising targets for therapy, as well as potential biomarkers for disease. T cells from patients with SLE express decreased levels of the T cell receptor (TCR) - associated CD3 zeta (ζ) chain, a feature directly linked to their poor interleukin (IL)-2 production. We recently showed that the splicing regulator alternative splicing factor/ splicing factor 2 (ASF/SF2) enhances the expression of CD3ζ chain by limiting the production of an unstable mRNA splice variant. Here, we show that ASF/SF2 expression levels are decreased in T cells from SLE patients, and correlate with CD3ζ chain expression. Moreover, ASF/SF2 expression correlates inversely with SLE disease activity index (SLEDAI). Further, replenishment of ASF/SF2 in SLE T cells restores IL2 production. These results identify ASF/SF2 as a novel regulator of IL2 expression in human T cells and a potential molecular mechanism underlying the altered T cell defect in SLE.

A New Cellular Weapon to Kill Leukaemic B-Cells = سلاح خلوي جديد لقتل خلايا ( ب ) المسببة لإبيضاض الدم

A New Cellular Weapon to Kill Leukaemic B-Cells = سلاح خلوي جديد لقتل خلايا ( ب ) المسببة لإبيضاض الدم

CD27 costimulation augments the survival and antitumor activity of redirected human T cells in vivo

AbstractThe costimulatory effects of CD27 on T lymphocyte effector function and memory formation has been confined to evaluations in mouse models, in vitro human cell culture systems, and clinical observations. Here, we tested whether CD27 costimulation actively enhances human T-cell function, expansion, and survival in vitro and in vivo. Human T cells transduced to express an antigen-specific chimeric antigen receptor (CAR-T) containing an intracellular CD3 zeta (CD3ζ) chain signaling module with the CD27 costimulatory motif in tandem exerted increased antigen-stimulated effector functions in vitro, including cytokine secretion and cytotoxicity, compared with CAR-T with CD3ζ alone. After antigen stimulation in vitro, CD27-bearing CAR-T cells also proliferated, up-regulated Bcl-XL protein expression, resisted apoptosis, and underwent increased numerical expansion. The greatest impact of CD27 was noted in vivo, where transferred CAR-T cells with CD27 demonstrated heightened persistence after infusion, facilitating improved regression of human cancer in a xenogeneic allograft model. This tumor regression was similar to that achieved with CD28- or 4-1BB–costimulated CARs, and heightened persistence was similar to 4-1BB but greater than CD28. Thus, CD27 costimulation enhances expansion, effector function, and survival of human CAR-T cells in vitro and augments human T-cell persistence and antitumor activity in vivo.

JAK3, STAT3 and CD3-zeta Signaling Proteins Status in Regard to the Lymphocytes Function in Patients with Ovarian Cancer

Several groups of author have published that, in most cases of carcinoma, circulating lymphocytes are unable to carry out immune functions successfully. A molecular mechanism responsible for T lymphocytes defective reactivity in cancer patients is not completely defined. We evaluated whether the impaired function of peripheral blood lymphocytes (PBLs) from ovarian cancer patients could be associated with signaling elements such as JAK3, STAT3 and CD3-zeta chain. The study addressed to the simultaneous expression and phosphorylation status of mentioned molecules evaluation in regard to lymphocyte function in patients with advanced ovarian cancer has not yet been demonstrated by others. We found that PBLs of cancer patients showed lower JAK3, CD3-zeta molecules expression levels, as well as lower STAT3 and CD3-zeta phosphorylation levels than cells of control. The lower proliferative response and IL-2 production capacity of cancer patients PBLs in comparison with that of the control group cells were the functional consequences of reported in this study signaling abnormalities.

Vaccination‐induced functional competence of circulating human tumor‐specific CD8 T‐cells

T-cells specific for foreign (e.g., viral) antigens can give rise to strong protective immune responses, whereas self/tumor antigen-specific T-cells are thought to be less powerful. However, synthetic T-cell vaccines composed of Melan-A/MART-1 peptide, CpG and IFA can induce high frequencies of tumor-specific CD8 T-cells in PBMC of melanoma patients. Here we analyzed the functionality of these T-cells directly ex vivo, by multiparameter flow cytometry. The production of multiple cytokines (IFNγ, TNFα, IL-2) and upregulation of LAMP-1 (CD107a) by tumor (Melan-A/MART-1) specific T-cells was comparable to virus (EBV-BMLF1) specific CD8 T-cells. Furthermore, phosphorylation of STAT1, STAT5 and ERK1/2, and expression of CD3 zeta chain were similar in tumor- and virus-specific T-cells, demonstrating functional signaling pathways. Interestingly, high frequencies of functionally competent T-cells were induced irrespective of patient's age or gender. Finally, CD8 T-cell function correlated with disease-free survival. However, this result is preliminary since the study was a Phase I clinical trial. We conclude that human tumor-specific CD8 T-cells can reach functional competence in vivo, encouraging further development and Phase III trials assessing the clinical efficacy of robust vaccination strategies.

Down-regulation of CD3zeta is a breast cancer biomarker associated with immune suppression

This work was done to establish a correlation between serum protein expression profiles and breast cancer. A high-density antibody microarray was used to identify serum protein expression profiles. Proteins up- or down-regulated by 2-fold compared with normal controls were used for hierarchical clustering analysis. ELISA and immunohistochemistry were used for validation of protein array data. The CD3zeta chain was one of the down-regulated molecules identified by the antibody array analysis and confirmed by ELISA and immunohistochemistry. The mean CD3zeta levels in breast cancer and in normal control were 119±23 and 156±35 ng/ml, respectively. The AUC (area under the curve) of CD3zeta was 0.837±0.057. Forty-one out of 65 breast cancer cases (63.08%) showed reduced CD3zeta expression on tumour-infiltrating lymphocytes by immunohistochemistry. This is the first report describing down-regulation of CD3zeta expression on breast cancer for the Chinese population and suggests that an immunosuppressive status plays an important role in breast cancer progression.

Retrovirally engineered T‐cell‐based immunotherapy targeting type III variant epidermal growth factor receptor, a glioma‐associated antigen

The isotype of epidermal growth factor receptor variant III (EGFRvIII) is often identified in glioblastomas. Previously, we created a mouse monoclonal antibody, 3C10 (IgG2b), that specifically recognized EGFRvIII, and a recombinant single-chain variable fragment of 3C10. The aim of the current study was to develop genetically engineered T cells, termed T-bodies, that express a chimeric receptor consisting of the 3C10 single-chain variable fragment coupled to signaling modules such as the CD3zeta (ζ) chain, for the treatment of tumors expressing mutant EGFR. After successful construction of the chimeric 3C10/CD3ζ T-cell receptor, its expression on the T-body was observed using western blotting and flow cytometry. The specificity of the T-body for EGFRvIII was evaluated using an interferon-gamma Elispot assay and a standard (51) Cr-release cytotoxicity assay. Furthermore, we demonstrated that the systemically delivered T-body infiltrated the intrabrain tumor and significantly delayed tumor growth. These results indicate that the T-body expressing the chimeric 3C10/CD3ζ T-cell receptor specifically recognized glioma cells expressing EGFRvIII. In conclusion, T-body-based immunotherapy appears to be a promising approach for the treatment of glioma.

Effect of L‐arginine supplementation on immune responsiveness in patients with sickle cell disease

L-arginine (L-Arg) is deficient in sickle cell disease (SSD) during vasoocclusion. We investigated possible causal relationship between L-Arg deficiency and immune dysfunction in SSD in steady-state. Fifteen patients with SSD in steady-state and 13 controls were studied. Plasma L-Arg levels were measured using liquid chromatography. T cell subsets and CD3zeta (CD3zeta) chain expression were analyzed using flow cytometry. Lymphocyte proliferative response to phytohemagglutinin (PHA) and production of IL-6 and interferon-gamma (IFN-gamma) were evaluated with and without L-Arg. SSD patients had significantly lower L-Arg levels than controls. CD3 and CD19 cell populations were comparable for both groups, but SSD patients had above normal numbers of natural killer cells (P = 0.06). Patients and controls exhibited significantly increased lymphocyte blastogenesis to PHA after introduction of L-Arg to cultures; response of patients was significantly greater than values for control individuals. Proliferative response to candida in SSD patients was significantly lower than in controls; L-Arg supplementation did not increase this response. L-Arg had no effect on blastogenic response to PPD and candida albicans. No effect was likewise seen in production of IL-6 and IFN-gamma after addition of L-Arg. CD3zeta chain expression increased after addition of L-Arg in both groups; differences were insignificant. L-Arg levels in steady-state SSD are significantly lower than in controls. L-Arg supplementation enhanced lymphocyte blastogenesis to PHA for both controls and patients, but not in response to antigen. There were no significant differences in CD3zeta chain expression although upregulation of expression occurred after L-Arg supplementation for both groups.

The splicing factor ASF/SF2 regulates the expression of the human T cell receptor CD3 zeta chain (143.11)

Abstract T cells from patients with systemic lupus erythematosus (SLE) express decreased levels of the T cell receptor associated (TCR)-associated CD3 zeta (ζ) chain, a feature directly linked to their aberrant function. The decrease in CD3ζ protein expression is in part due to decreased levels of the functional wild-type (WT) isoform of the 3` untranslated region (UTR) of CD3ζ mRNA with concomitant increased levels of an unstable alternatively spliced (AS) isoform. In order to identify factors involved in the post transcriptional regulation of CD3ζ, we performed mass spectrometric analysis of Jurkat T cell nuclear proteins “pulled down” by a CD3ζ 3`UTR defined oligonucleotide which identified the splicing protein Alternative Splicing factor / Splicing factor 2 (ASF/SF2). We show for the first time that ASF/SF2 binds specifically to the 3`UTR of CD3ζ and regulates expression of CD3ζ protein by limiting the production of the alternatively spliced isoform. During activation of human T cells, an increase in the WT CD3ζ mRNA is associated with increased expression of ASF/SF2. Finally, we show a significant correlation between ASF/SF2 and CD3ζ protein levels in T cells from SLE patients. These results identify ASF/SF2 as a novel factor in the regulation of CD3ζ expression in human T cells, and a potential molecular mechanism underlying the altered CD3ζ chain expression in SLE.

4-1BB and CD28 Signaling Plays a Synergistic Role in Redirecting Umbilical Cord Blood T Cells Against B-Cell Malignancies

Umbilical cord blood (UCB) T cells can be redirected to kill leukemia and lymphoma cells by engineering with a single-chain chimeric antigen receptor (CAR) and thus may have general applications in adoptive cell therapy. However, the role of costimulatory molecules in UCB T-cell activation and effector functions in context with CAR remains elusive. To investigate the effect of costimulatory molecules (4-1BB and CD28) on UCB T cells, we transduced UCB T cells with lentiviral vectors expressing Green Fluorescent Protein (GFP) and CAR for CD19 containing an intracellular domain of the CD3zeta chain and either a 4-1BB (UCB-19BBzeta) or a CD28 intracellular domain (UCB-1928zeta), both (UCB-1928BBzeta), or neither (UCB-19zeta). We found that UCB-19BBzeta and UCB-28BBzeta T cells exhibited more cytotoxicity to CD19(+) leukemia and lymphoma cell lines than UCB-19zeta and UCB-1928zeta, although differences in secretion of interleukin-2 and interferon-gamma by these T cells were not evident. In vivo adoptive transfer of these T cells into intraperitoneal tumor-bearing mice demonstrated that UCB-19BBzeta and UCB-1928BBzeta T cells mounted the most potent antitumor response. The mice adoptively transferred with UCB-1928BBzeta cells survived longer than the mice with UCB-19BBzeta. Moreover, UCB-1928BBzeta T cells mounted a more robust antitumor response than UCB-19BBzeta in a systemic tumor model. Our data suggest a synergistic role of 4-1BB and CD28 costimulation in engineering antileukemia UCB effector cells and implicate a design for redirected UCB T-cell therapy for refractory leukemia.

CD8+ T Cells Engineered to Express a ROR1-Specific Chimeric Antigen Receptor Specifically Recognize ROR1 Positive B Cell Tumors.

Abstract 930The orphan tyrosine kinase receptor ROR1 was previously identified as a highly expressed gene by expression profiling of B cell chronic lymphocytic leukemia (B-CLL), [Klein et al. J Exp Med 2001], and has subsequently been shown to be expressed on mantle cell lymphoma (MCL) and a subset of B cell acute lymphoblastic leukemias (B-ALL). ROR1 encodes a 105 kDa protein that contains Ig-like, cysteine rich, kringle, tyrosine kinase and proline rich domains and is expressed during embryonic development but is absent on normal adult tissues including non-malignant B cells. The function of ROR1 in normal and malignant cells is not known, although secreted Wnt proteins have been proposed as candidate ligands. Analysis of ROR1 protein expression using specific polyclonal antibodies revealed uniform, stable, and restricted cell surface expression on B-CLL, suggesting this molecule is a candidate for targeted immunotherapy of B cell malignancies [Baskar et al. Clin Cancer Res 2008]. We constructed a lentiviral vector that encodes a chimeric antigen receptor (CAR) consisting of single chain variable (scFV) fragments of the heavy and light chains of a murine monoclonal antibody specific for ROR1, linked to an IgG4 Fc domain, the T cell receptor CD3 zeta chain and a CD28 costimulatory domain. The specificity and function of the ROR1 CAR was compared with a similarly designed CAR specific for the CD20 molecule, which is expressed on both malignant and normal B cells, and is being targeted with gene-modified T cells in clinical trials. Primary human CD8+ T cells were transduced with the ROR1 and CD20-specific CARs respectively, and T cells expressing high levels of the receptors were sort-purified using an anti-Fc antibody. T cells that expressed either the ROR1-specific CAR or the CD20-specific CAR efficiently lysed primary B-CLL samples (5/5) obtained from patients with advanced disease, and also lysed a MCL cell line (JeKo-1), and a ROR1+ B-ALL cell line (BALL-1). ROR1-specific T cells did not recognize the myeloid leukemia cell line K562, but efficiently lysed K562 cells that had been transfected to express ROR1, confirming the specific recognition of ROR1 on target cells. Consistent with the expression pattern of the target molecules, T cells that expressed the CD20-specific CAR also efficiently lysed normal primary and EBV-transformed B cells, but T cells that expressed the ROR1-specific CAR did not recognize nonmalignant or EBV-transformed B cells. Activation of normal B cells by engagement of the B cell receptor or activation through CD40 induced B cell proliferation and upregulation of the CD80 and CD86 costimulatory molecules, but did not result in ROR1 surface expression by flow cytometry or recognition by T cells that expressed the ROR1-specific CAR. These results suggest that targeting ROR1 with gene-modified T cells may have advantages over targeting B cell-lineage restricted molecules such as CD19 and CD20 that are expressed on normal mature B cells. Studies to determine whether ROR1 is expressed during a stage of normal B cell development are in progress. ROR1 is highly conserved in non-human primates and this model may be suitable to determine potential toxicities of adoptive immunotherapy with ROR1-specific CAR expressing T cells. Disclosures:No relevant conflicts of interest to declare.

Evaluation of fall versus spring dormant planting of hardwood willow cuttings with and without soaking treatment

The enzyme telomerase is essential for maintaining the replicative capacity of memory T cells. Although CD28 costimulatory signals can up-regulate telomerase activity, human CD8⁺ T cells lose CD28 expression after repeated activation. Nevertheless, telomerase is still inducible in CD8⁺CD28⁻ T cells. To identify alternative costimulatory pathways that may be involved, we introduced chimeric receptors containing the signaling domains of CD28, CD27, CD137, CD134, and ICOS in series with the CD3 zeta (ζ) chain into primary human CD8⁺ T cells. Although CD3 ζ-chain signals alone were ineffective, triggering of all the other constructs induced proliferation and telomerase activity. However, not all CD8⁺CD28⁻ T cells could up-regulate this enzyme. The further fractionation of CD8⁺CD28⁻ T cells into CD8⁺CD28⁻ CD27⁺ and CD8⁺CD28⁻CD27⁻ subsets showed that the latter had significantly shorter telomeres and extremely poor telomerase activity. The restoration of CD28 signaling in CD8⁺CD28⁻CD27⁻ T cells could not reverse the low telomerase activity that was not due to decreased expression of human telomerase reverse transcriptase, the enzyme catalytic subunit. Instead, the defect was associated with decreased phosphorylation of the kinase Akt, that phosphorylates human telomerase reverse transcriptase to induce telomerase activity. Furthermore, the defective Akt phosphorylation in these cells was specific for the Ser⁴⁷³ but not the Thr³⁰⁸ phosphorylation site of this molecule. Telomerase down-regulation in highly differentiated CD8⁺CD28⁻CD27⁻ T cells marks their inexorable progress toward a replicative end stage after activation. This limits the ability of memory CD8⁺ T cells to be maintained by continuous proliferation in vivo.

Activated CD11b+CD15+Granulocytes Increase in the Blood of Patients with Uveal Melanoma

To determine whether activated CD11b(+) CD15(+) granulocytes increase in the blood of patients with uveal melanoma. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation from the blood of patients with primary choroidal/ciliochoroidal uveal melanomas (six women, four men; age range, 46-91 years) and healthy control donors (14 women, 10 men; age range, 50-81 years). The expression of CD15 and CD68 on CD11b(+) myeloid cells within PBMCs and primary uveal melanomas was evaluated by flow cytometry. CD3zeta chain expression by CD3epsilon(+) T cells in PBMCs and within primary uveal melanomas was measured as an indirect indication of T-cell function. The percentage of CD11b(+) cells in PBMCs of patients with uveal melanoma increased 1.8-fold in comparison to healthy donors and comprised three subsets: CD68 negative CD15(+) granulocytes, which increased 4.1-fold; CD68(-) CD15(-) cells, which increased threefold; and CD68(+) CD15(low) cells, which were unchanged. A significant (2.7-fold) reduction in CD3zeta chain expression on CD3epsilon(+) T cells, a marker of T-cell dysfunction, was observed in PBMCs of patients with uveal melanoma in comparison with healthy control subjects and correlated significantly with the percentage of CD11b(+) cells in PBMCs. CD3zeta chain expression on T cells within primary tumors was equivalent to CD3zeta expression in PBMCs of the same patient in four of five patients analyzed. Activated CD11b(+) CD15(+) granulocytes expand in the blood of patients with uveal melanoma and may contribute to immune evasion by ocular tumors by inhibiting T-cell function via decreasing CD3zeta chain expression.

Construction and Pre-Clinical Evaluation of An Anti-CD19 Chimeric Antigen Receptor

Adoptive T cell immunotherapy can cause regression of established malignancy. One promising approach is to transfer genes encoding chimeric antigen receptors (CARs) that specifically recognize tumor-associated antigens to T cells before the T cells are adoptively transferred to patients. We have constructed a CAR that consists of an anti-CD19 single chain variable region (scFv) that is coupled to a portion of the CD28 costimulatory molecule and the signaling component of the CD3-zeta chain. CD19 is a promising target for immunotherapy because most malignant B cell express CD19, but the only normal cells that express CD19 are B cells, B cell precursors, and perhaps follicular dendritic cells. We have demonstrated that gamma-retroviruses encoding the anti-CD19 CAR can be used to transduce human T cells and that these transduced T cells specifically recognize CD19+ targets. To transduce T cells, we stimulated T cells with the anti-CD3 monoclonal antibody OKT3 on day 0 then conducted sequential retroviral transductions on day 2 and on day 3. Transductions were performed by spin-loading retroviruses onto RetroNectin (Takara) coated culture plates followed by overnight incubation of the OKT3- stimulated T cells on the plates. Forty-five to sixty-seven percent of T cells expressed the anti-CD19 CAR as measured by flow cytometry 7–8 days after transduction (n=8). Anti-CD19-CAR-transduced CD8+ and CD4+ T cells produced IFNg and IL-2 specifically in response to stimulation with CD19+ target cells. The transduced T cells specifically killed primary chronic lymphocytic leukemia (CLL) cells. T cells from CLL patients that were either untreated or previously treated with fludarabine plus rituximab could be transduced and induced to proliferate sufficiently to provide enough cells for clinical adoptive T cell transfer. In addition, we adapted this protocol for use in CLL patients with very high peripheral blood leukemia cell counts by depleting CD19+ cells using magnetic bead sorting prior to OKT3 stimulation. In preparation for a clinical trial that will enroll patients with advanced B cell malignancies, we have generated a producer cell clone that produces GALV (Gibbon ape leukemia virus)-enveloped gamma-retroviruses encoding the anti-CD19 CAR, and we have produced sufficient retroviral supernatant for the proposed clinical trial under good manufacturing practice (GMP) conditions.

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.

Chimeric NKG2D receptor–expressing T cells as an immunotherapy for multiple myeloma

Most myeloma tumor cells from patients express NKG2D ligands. We have reported the development of a chimeric NKG2D receptor (chNKG2D), which consists of the NKG2D receptor fused to the CD3zeta chain. T cells expressing this receptor kill and produce cytokines in response to NKG2D-ligand+ tumor cells. Therefore, we investigated whether human chNKG2D T cells respond against human myeloma cells. ChNKG2D T cells were generated from healthy donors and myeloma patients. The effector phase of chNKG2D T cells was analyzed by cell-surface marker expression and human myeloma cell lines were tested for expression of NKG2D ligands. Lysis of myeloma cell lines and cytokine secretion by chNKG2D T cells was determined. ChNKG2D T cells grown in serum-free media, or cyropreserved, were assessed for effector cell functions. Myeloma cell lines expressed NKG2D ligands. ChNKG2D T cells from healthy donors and myeloma patients lysed myeloma cells, and secreted proinflammatory cytokines when cultured with myeloma cells or patient bone marrow, but not with peripheral blood mononuclear cells or normal bone marrow. Lysis of myeloma cells was dependent on chNKG2D T-cell expression of NKG2D and perforin. Additionally, chNKG2D T cells upregulated CD45RO, did not express CD57, and maintained expression of CD27, CD62L, and CCR7, indicating that the T cells were at an early effector stage. Finally, we showed that chNKG2D T cells generated with serum-free media, or when cryopreserved, maintained effector functions. ChNKG2D T cells respond to human myeloma cells and can be generated using clinically applicable cell culture techniques.

The RNA-stabilizing Protein HuR Regulates the Expression of ζ Chain of the Human T Cell Receptor-associated CD3 Complex

T cell dysfunction is crucial to the pathogenesis of systemic lupus erythematosus (SLE); however, the molecular mechanisms involved in the deficient expression of the T cell receptor-associated CD3zeta chain in SLE are not clear. SLE T cells express abnormally increased levels of an alternatively spliced isoform of CD3zeta that lacks a 562-bp region in its 3'-untranslated region (UTR). We showed previously that two adenosine/uridine-rich elements (ARE) in this splice-deleted region of CD3zeta transcript are critical for the mRNA stability and protein expression of CD3zeta. In this study we show for the first time that the mRNA-stabilizing protein HuR binds to these two ARE bearing regions of CD3zeta 3'-UTR. Knockdown of HuR resulted in decreased expression of the CD3zeta chain, whereas overexpression led to the increase of CD3zeta chain levels. Additionally, overexpression of HuR in human T cells resulted in increased mRNA stability of CD3zeta. Our results identify the 3'-UTR of CD3zeta as a novel target for the mRNA-stabilizing protein HuR. Thus, the absence of two critical AREs in the alternatively spliced CD3zeta 3'-UTR found in SLE T cells may result in decreased HuR binding, representing a possible molecular mechanism contributing to the reduced stability and expression of CD3zeta in SLE.

A Lysosomal Protein Negatively Regulates Surface T Cell Antigen Receptor Expression by Promoting CD3ζ-Chain Degradation

Modulation of surface T cell antigen receptor (TCR) expression is an important mechanism for the regulation of immune responses and the prevention of T cell hyperactivation and autoimmunity. The TCR is rapidly internalized after antigen stimulation and then degraded in lysosomes. However, few of the molecules involved in this process have been identified. We demonstrate that the lysosomal protein LAPTM5 negatively regulated surface TCR expression by specifically interacting with the invariant signal-transducing CD3zeta chain and promoting its degradation without affecting other CD3 proteins, CD3epsilon, CD3delta, or CD3gamma. TCR downmodulation required the polyproline-tyrosine motifs and the ubiquitin-interacting motif of LAPTM5. LAPTM5 deficiency resulted in elevated TCR expression on both CD4(+)CD8(+) thymocytes and spleen T cells after CD3 stimulation, as well as enhanced T cell responses in vitro and in vivo. These results identify a lysosomal protein important for CD3zeta degradation and illustrate a unique mechanism for the control of surface TCR expression and T cell activation.

Alterations in the expression of signal-transducing CD3ζ chain in T cells from patients with chronic inflammatory/autoimmune diseases

The CD3 zeta chain, a component of the T cell receptor (TCR)/CD3 complex, is considered to be a limiting factor in the assembly and transport of the TCR/CD3 complex to the cell surface and is crucial to receptor signaling function. Recent studies have demonstrated altered expression and function of this signal transduction molecule in T and natural killer cells in patients with chronic inflammatory/autoimmune diseases. In this review, current knowledge concerning the expression of CD3 zeta chain as well as the mechanisms responsible for abnormal expression of this molecule in systemic lupus erythematosus, rheumatoid arthritis, and childhood idiopathic nephrotic syndrome are summarized.