T-cell Receptor Zeta Chain Research Articles

RNA-Sequencing Analysis and Expression of Lymphocyte-Specific Protein Tyrosine Kinase, Linker for Activation of T Cells, and 70-kDa T-Cell Receptor Zeta-Chain Associated Protein Kinase in Rat Liver Transplant Rejection.

The prevention and treatment of liver transplant rejection remain challenging. We investigated the pathophysiological mechanisms of liver transplant rejection in rats and screened candidate genes to determine their degree of rejection response for possible development of potential therapeutic targets. Brown Norway-Brown Norway transplant tolerant models and Lewis-Brown Norway transplant rejection models were established. We collected liver tissue and venous blood at 7 days posttransplant for hematoxylin and eosin staining and RNA sequencing analysis, respectively. We conducted differential expression gene analysis, KEGG and GO enrichment analysis. We performed immunohistochemistry to detect highly expressed immunerelated proteins, including lymphocyte-specific protein tyrosine kinase, linker for activation of T cells, and 70-kDa T-cell receptor zeta-chain-associated protein kinase. Significant differences were found in liver function and Banff scores between rejection and tolerant groups, indicating the successful establishment of liver transplant models. RNA-sequencing screened 7521 differentially expressed genes, with 3355 upregulated and 3058 downregulated. KEGG analysis of upregulated genes showed that 8 of the top 20 enrichment pathways were associated with immune system processes and 5 were related to immune system diseases. Among these immune pathways, 289 genes were upregulated; of these, 147 genes were removed after comparison with the IMMPORT database, of which 97 genes were significantly changed. Our GO analysis showed upregulated genes mainly participating in immune response processes, with downregulated genes mainly participating in metabolic processes. Real-time polymerase chain reaction and immunohistochemistry verified expression of the immune-related proteins, consistent with RNAsequencing results, which were mainly expressed in inflammatory cells in sinus and portal vein. Immune-related genes were found to be associated with liver transplant rejection. The 3 immune-related genes that we analyzed may play a role in liver transplant rejection and can possibly serve as candidate markers for monitoring the degree of liver transplant rejection.

Targeting Primary Pre-B Cell Acute Lymphoblastic Leukemia and CD19-Negative Relapses Using Trivalent CAR T Cells

B-Acute Lymphoblastic Leukemia (B-ALL) is the most common malignancy in children, and limited treatment options exist for patients with relapsed or refractory disease. Cellular immunotherapy, specifically chimeric antigen receptor (CAR) T cells targeting CD19, have demonstrated remarkable efficacy in treating B-ALL. However, recent reports show that up to 40% of patients who relapse after CD19 CAR T cell therapy have CD19-negative disease, justifying a need to expand CAR T cell therapy for B-ALL to include additional tumor-associated antigens.Here, we hypothesize that targeting three distinct leukemia antigens including CD19, CD20, and CD22 will improve B-ALL therapy outcomes and control disease progression during CD19-negative relapse.We designed two trivalent CAR T cell products with exodomains derived from single chain variable fragments (ScFv) targeting CD19 (FMC63 ScFv), CD20 (Rituximab ScFv), and CD22 (m971 ScFv). Using viral 2A intervening sequences for near equal expression, the first T cell product expresses the three CARs individually on the surface of a single T cell (TriCAR), and the second T cell product expresses a traditional single CAR targeting CD19 and a second bi-specific CAR targeting CD20 and CD22 through a tandem arrangement (SideCAR). All CARs are fused to the intracellular signaling domains of the co-stimulatory molecule 4-1BB and the T-cell receptor zeta (ζ) chain (2nd generation). Donor T cells were successfully engineered to express the CARs using a retroviral system and the surface expression of these CAR molecules was confirmed by flow cytometry. Using a target expression validated panel of patient derived B-ALL cells (US7 CD19/CD20/CD22 +++/++/++, LAX-56 +++/+/+, TXL-2 +++/++/+++), we observed that TriCAR and SideCAR T cells killed ALL cells more robustly than CD19 CAR T cells at low effector to target (E:T) ratios. TriCAR and SideCAR T cells secreted similar levels of IFN-γ/TNF-α when compared to CD19 CAR T cells suggesting a safety profile similar to the CD19 CAR T cells, but with enhanced killing. Further, we tested the efficacy of TriCAR and SideCAR T cells against primary CD19-negative relapsed bone marrow samples and CRISPR CD19 knockouts of the three primary ALL samples. Using these models of CD19 escape we demonstrated that trivalent CAR T cells effectively mitigated CD19 negative relapse, producing IFN-γ and TNF-α and killing CD19-negative primary ALL, while CD19 CAR T cells remained ineffective. Finally, we interrogated immune synapse (IS) microcluster formation and actin dynamics during interactions between CAR T cells and primary B-ALL cells by quantitative imaging flow cytometry. All CAR T cells exhibited higher actin polymerization compared to non-transduced T cell controls. TriCAR T cells formed significantly higher number of IS microclusters with different morphologies of actin polymerization compared to CD19 CAR T cells, suggesting distinct remodeling and dynamics of T cell polarization and immunoactivity, when interacting with TXL-2 primary B-ALL cells. TriCAR T cells formed IS microclusters, while CD19 CAR T cells failed to form IS with CD19 knockout TXL-2 cells.In conclusion, trivalent CAR T cells are effective at targeting primary ALL cells with varying antigen profiles and at mitigating CD19-negative relapse. This strategy has the potential for use as a front-line therapy for primary ALL as well as a salvage therapy for patients with CD19-negative disease relapse. DisclosuresNo relevant conflicts of interest to declare.

Abstract 6605: Functional screening of bispecific CAR ectodomains targeting HER2 and GD2 in melanoma

Abstract We describe the development of targeted immunotherapy using a bispecific chimeric antigen receptor (CAR) modified T-cell directed against the tumor associated antigens, human epidermal growth factor receptor 2 (HER2) and the disialoganglioside GD2, surface expressed in a large proportion of melanoma. Melanoma remains the most aggressive of skin cancers with a 5-year survival rate less than 20% in those with metastatic disease. For these patients, immunotherapy has offered an alternative promising approach in the recent years. We designed bispecific CAR molecule ectodomains (n=8) using different permutations of single chain variable fragments (scFv) recognizing HER2 and GD2 in tandem arrangements. HER2 recognizing scFvs were derived from FRP5 and 4D5 (Trastuzumab), and GD2 recognizing scFvs were derived from 14G2a and humanized 3F8. The ectodomain containing the tandem arrangements of two distinct scFvs was joined to a signaling endodomain of the co-stimulatory molecules, CD28 and OX40, and a T-cell receptor zeta-chain. Primary human T cells were successfully transduced using a retroviral system achieving 37-73% efficiency of surface expression among various bispecific CAR constructs, as determined by flow cytometry. The function of bispecific CAR T cells were evaluated in a series of immunoassays against melanoma cell lines with variable levels of HER2 and GD2 expression. T cells expressing CARs with single specificity (HER2 or GD2) and non-transduced T cells from the same donor were used as controls. T cells transduced with bispecific TanCAR molecules proliferated after co-culture with melanoma cells. Upon evaluation of the cytokine release after encountering melanoma cells, 14G2a/FRP5 derived bispecific CAR T cells demonstrated the highest level of interferon-γ release with 14G2a-FRP5 (distal-proximal) tandem arrangement exhibiting the most robust activation. Further, consistent cytotoxicity against melanoma cells was observed in 4-hour 51Cr-release assay across donors for T cells expressing CAR ectodomain with 4D5-14G2a, 14G2a-FRP5, FRP5-14G2a and hu3F8-FRP5 tandem arrangements. Of these, T cells expressing 14G2a-FRP5, FRP5-14G2a and hu3F8-FRP5 tandem ectodomain arrangements demonstrated long-term tumor control in an impedance based in vitro tumor cell killing assay. In conclusion, we have rationally designed and generated HER2/GD2 bispecific CAR T cells with the intent of targeting the antigenic heterogeneity in melanoma. We have successfully screened multiple iterations of bispecific CAR ectodomains against melanoma demonstrating that the scFv as well as their sequence of tandem arrangement influences the antitumor function of modified T cells. The antitumor function of the top three candidate bispecific ectodomains identified will be further evaluated in pre-clinical animal models to determine the optimal construct for clinical development. Citation Format: Sujith K. Joseph, Shoba A. Navai, Zakaria Grada, Kristen Fousek, Pretty R. Mathew, Ankita Shree, Amanda Wakefield, Nabil Ahmed, Meenakshi Hegde. Functional screening of bispecific CAR ectodomains targeting HER2 and GD2 in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6605.

Chimeric Antigen Receptor Signaling Domains Differentially Regulate Proliferation and Native T Cell Receptor Function in Virus-Specific T Cells.

The efficacy of T cells expressing chimeric antigen receptors (CARs) for solid tumors has been limited by insufficient CAR T cell expansion and persistence. The use of virus-specific T cells (VSTs) as carriers for CARs may overcome this limitation since CAR-VSTs can be boosted by viral vaccines or oncolytic viruses. However, there is limited understanding of the optimal combination of endodomains and their influence on the native T cell receptor (TCR) in VSTs. We therefore compared the function of GD2.CARs expressing the TCR zeta chain (ζ) alone or combined with endodomains from CD28 and 4-1BB in varicella zoster virus-specific (VZV) T cells. VZVSTs expressing GD2-CARs recognized VZV-derived peptides and killed GD2-expressing tumor cells. However, after repeated stimulation through their native TCR, the expansion of GD2-CAR.CD28ζ-VZVSTs was 3.3-fold greater (p < 0.001) than non-transduced VZVSTs, whereas GD2-CARζ- and GD2-CAR.41BBζ inhibited VZVST expansion (p < 0.01). Compared to control VZVSTs, GD2-CAR.ζ VZVSTs showed a greater frequency of apoptotic (p < 0.01) T cells, whereas prolonged downregulation of the native αβ TCR was observed in GD2-CAR.41BBζ VZVSTs (p < 0.001). We confirmed that CD28ζ can best maintain TCR function by expressing GD2.CARs in Epstein-Barr virus-specific T cells and CD19-CARs in VZVSTs. In response to CAR stimulation VSTs with CD28ζ endodomains also showed the greatest expansion (6 fold > GD2-CAR.41BBζ VZVSTs (p < 0.001), however anti-tumor efficacy was superior in GD2-CAR.41BBζ-VZVSTs. These findings demonstrate that CAR signaling domains can enhance or diminish the function of the native TCR and indicate that only CD28ζ may preserve the function of the native TCR in tonically signaling CAR-VSTs.

Abstract A50: Trivalent CAR T cells mitigate CD19-negative relapse and improve tumor control in primary pre-B cell acute lymphoblastic leukemia (B-ALL)

Abstract B-acute lymphoblastic leukemia (B-ALL) is the most common malignancy in children, and limited treatment options exist for patients with relapsed or refractory disease. Cellular immunotherapy, specifically chimeric antigen receptor (CAR) T cells targeting CD19 have demonstrated remarkable efficacy in treating B-ALL. However, recent reports show that up to 40% of patients who relapse after CD19 CAR T cell therapy have CD19-negative disease, justifying a need to expand CAR T cell therapy for B-ALL to include additional tumor-associated antigens (TAAs). Here, we hypothesize that targeting three distinct leukemia antigens CD19, CD20, and CD22 will improve B-ALL therapy outcomes and control disease progression during CD19-negative relapse. We designed two trivalent CAR T cell products with exodomains derived from single chain variable fragments (ScFv) targeting CD19 (FMC63 ScFv), CD20 (Rituximab ScFv), and CD22 (m971 ScFv) fused to the intracellular signaling domains of the co-stimulatory molecule 4-1BB and the T-cell receptor zeta chain (2nd generation). Using viral 2A intervening sequences for near equal expression, the first T cell product expresses the three CARs individually on the surface of a single T cell (TriCAR). The second T cell product expresses a traditional single CAR targeting CD19 and a second bi-specific CAR targeting CD20 and CD22 through a tandem arrangement (SideCAR). Donor T cells were successfully engineered to express the CARs using a retroviral system and the surface expression of these CAR molecules was confirmed by flow cytometry. Using a target expression validated panel of patient derived B-ALL cells (US7 CD19/CD20/CD22 +++/++/++, LAX-56 +++/+/+, TXL-2 +++/++/+++), we observed that TriCAR and SideCAR T cells killed ALL cells more robustly than CD19 CAR T cells at low effector to target ratios (E:T) in a 51Cr release cytotoxicity assay. TriCAR and SideCAR T cells secreted similar levels of IFN-gamma; when compared to CD19 CAR T cells demonstrating a safety profile very similar to the CD19 CAR T cells, but with enhanced killing. Further, we tested the efficacy of TriCAR and SideCAR T cells against primary CD19-negative relapsed bone marrow samples and CRISPR CD19 knockouts of the three primary ALL samples. Using these models of CD19 escape we demonstrated that trivalent CAR T cells effectively mitigated CD19 negative relapse, producing IFN-gamma; and killing CD19-negative primary ALL, while CD19 CAR T cells remained ineffective. In conclusion, trivalent CAR T cells are effective at targeting primary ALL cells of varying antigen profiles and mitigating CD19 negative relapse. This strategy has potential for use as a front-line therapy for primary ALL as well as a salvage therapy for patients with CD19-negative disease relapse. Citation Format: Kristen Fousek, Junji Watanabe, Xingyue An, Ann George, Heba Samaha, Shoba Navai, Tiara T. Byrd, Jonathan Kirzner, Hye Na Kim, Albert Jang, Sujith Joseph, Matthew Baker, Meenakshi Hegde, Navin Varadarajan, Nora Heisterkamp, Hisham Abdel-Azim, Nabil Ahmed. Trivalent CAR T cells mitigate CD19-negative relapse and improve tumor control in primary pre-B cell acute lymphoblastic leukemia (B-ALL) [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A50.

Targeting CD19-negative relapsed B-acute lymphoblastic leukemia using trivalent CAR T cells.

121 Background: Chimeric antigen receptor (CAR) T cells targeting CD19 have shown remarkable efficacy in treating relapsed B cell acute lymphoblastic leukemia (B-ALL). However, recent reports show that up to 40% of patients who relapse after CD19 CAR T cell therapy have CD19-negative disease, justifying a need to expand CAR T cell therapy for B-ALL to include additional tumor-associated antigens. We hypothesize that targeting CD19, CD20, and CD22 will improve B-ALL therapy outcomes and control disease progression during CD19-negative relapse. Methods: We designed two trivalent CAR T cell products with exodomains derived from single chain variable fragments (ScFv) targeting CD19, CD20, and CD22. Each CAR contains the 4-1BB and T-cell receptor zeta chains. Donor T cells were engineered to express the CARs using a retroviral system. We used primary CD19-negative relapsed bone marrow samples and CRISPR CD19 knockouts of primary ALL to model CD19 escape and standard cytotoxicity and immune assays to evaluate anti-tumor efficacy. Results: Due to the use of viral 2A sequences we detected near equal expression of each CAR by flow cytometry. The first T cell product expresses three CARs individually (TriCAR), and the second expresses a single CAR targeting CD19 and a second bi-specific CAR targeting CD20 and CD22 via a tandem arrangement (SideCAR). Using primary B-ALL cells, we observed that TriCAR and SideCAR T cells killed ALL cells more robustly than CD19 CAR T cells at low E:T ratios. Further, in ImageStream analysis of single cell interactions between CAR T cells and primary B-ALL cells, TriCAR T cells exhibited increased actin polymerization compared to CD19 CAR T cells, suggesting remodeling and increased cell activation. Finally, in multiple models of CD19 escape in primary ALL, we showed that trivalent CAR T cells mitigated CD19 negative relapse, producing IFN-γ/TNF-α and killing CD19-negative primary ALL, while CD19 CAR T cells remained ineffective. Conclusions: Trivalent CAR T cells effectively target primary ALL cells with varying antigen profiles and mitigate CD19-negative relapse. This strategy has the potential for use as an initial CAR therapy in relapsed ALL or a salvage therapy for patients with CD19-negative disease.

Intracellular cytoplasm-specific delivery of SH3 and SH2 domains of SLAP inhibits TcR-mediated signaling

Signaling events triggered by T cell receptor (TcR) stimulation are important targets for the development of common therapeutics for various autoimmune diseases. SLAP is a negative regulator of TcR-mediated signaling cascade via targeting TcR zeta chain for degradation through recruiting the ubiquitin ligase c-Cbl. In this study, we generated a transducible form of SH3 and SH2 domains of SLAP (ctSLAPΔC) which can be specifically targeted to the cytoplasm of a cell. ctSLAPΔC inhibited tyrosine phosphorylation of signaling mediators such as ZAP-70 and LAT involved in T cell activation, and effectively suppressed transcriptional activity of NFAT and NFκB upon TcR stimulation. The transduced ctSLAPΔC in T cells blocked the secretion of T cell-specific cytokines such as IL-2, IFNγ, IL-17A, and IL-4 and induced the expression of CD69 and CD25 on effector T cells without influencing the cell viability. Inhibition of TcR-mediated signaling via SLAP blocked the differentiation of naïve T cells into Th1, Th2 or Treg cells with different sensitivity, suggesting that qualitative and quantitative intensity of TcR-mediated signaling in the context of polarizing cytokines environment may be a critical factor to determine the differentiation fate of naïve T cells. These results suggest that cytoplasm-specific transduction of the SH3 and SH2 domains of SLAP has a therapeutic potential of being an immunosuppressive reagent for the treatment of various autoimmune diseases.

Upregulated TCR Zeta and Improved the IFN-γ Secretion and Cytotoxicity in T Cell from Patients with AML after IL-2, IL-7 and IL-12 Stimulation

T cells from patients with acute myeloid leukemia (AML) display antigen receptor–mediated signaling aberrations associated with defective T cell receptor (TCR) zeta chain, a subunit of the TCR/CD3 complex. Up-regulate TCR signaling activation by TCR zeta recombinant vector transfection was showed to reverse IL-2 production and increased the activation in T cells from patients with chronic myeloid leukemia. This study was undertaken to explore the possibility that increased TCR zeta gene expression stimulated with immune cytokines may upregulate T cell receptor signaling activation in T cells from patients with AML. CD3+ T cells sorted from peripheral blood of 8 cases with AML were induced with different immune cytokines, including IL-2, IL-7, IL-12, anti-CD3 and anti-CD28 antibodies in vitro. The result showed that TCR zeta chain protein was significantly upregulated after stimulation with IL-2 + IL-7 at 72 hours (p<0.05) by western-blot analysis, the expression of Zap-70 which is TCR zeta-associated protein was increased apparently after stimulation by combined IL-7 and IL-12 at 72 hours (p<0.05). A higher level of interferon (IFN)-γ secretion was showed obviously with single stimulating factor or combination with different cytokines (p<0.05), especially in IL-12 + IL-7, CD3 + CD28 + IL-2 or CD3 + CD28 + IL-7 combination. In addition, the cytotoxicity of T cell from patients with AML was achieved after stimulation with different cytokines combinations at 7 days, especially in CD3 + CD28 + IL-2 group and CD3 + CD28 + IL-2 + IL-12 group. These result showed that downregulation of the TCR zeta chain in T cell from patients with AML is a reversible event. Combined different cytokines could restore TCR zeta chain deficiency and enhance IFN-γ production and cytotoxicity in T cells from AML patients. It is possible that upregulated TCR zeta chain expression in T cell from AML patients will effectively increase or recover their activation of antileukemia cytotoxicity. DisclosuresChen:National Natural Science Foundation of China (no. 81100353, 81270604), the Fundamental Research Funds for the Central Universities (No. 21611447, 21612116), And Medical Science Foundation of Guangdong Province(A2011325): Research Funding.

Abstract LB-199: A rationally designed bispecific chimeric antigen receptor molecule that simultaneously redirects T cells to target HER2 and GD2 in osteosarcoma

Abstract Tumor immunotherapy with chimeric antigen receptor (CAR)-modified T cells has shown substantial efficacy in early phase clinical trials. Targeting a single tumor antigen could however result in creation of antigen escape variants. One strategy to overcome this immune evasion mechanism is to simultaneously target two or more tumor restricted antigens (TAAs). We have created a novel bispecific CAR molecule that can effectively target HER2 and GD2 in osteosarcoma (OS), using in silico modeling. The extracellular domain of the bispecific CAR consists of two distinct antigen-recognition domains linked by a Gly-Ser linker; one for HER2 (scFv derived from monoclonal antibody FRP5) and the other for GD2 (scFv derived from monoclonal antibody 14g2a), joined in tandem (TanCAR). This extracellular-domain was assembled on Clone Manager®, then cloned into the Gateway® entry vector pDONR™221, sequence-verified and cloned inline with a signaling domain of the co-stimulatory molecule, CD28 and T-cell receptor zeta-chain. Healthy donor and patients’ T cells were transduced with HER2/GD2 TanCAR using a retroviral system. Surface expression of the TanCAR extracellular domain in its entirety was confirmed by flowcytometry using a HER2-scFv and another GD2-scFv specific method. We studied the varying expression pattern of HER2 and GD2 in 12 OS cell lines; e.g. LM7 was HER2/GD2 +/+, HOS HER2/GD2 +/- and IOR/OS9 was HER2/GD2 -/+. Burkitt's lymphoma cell line Raji, was used as a HER2/GD2 -/- control. TanCAR T cells released immunostimulatory cytokines (IL-2, IFN-γ) in co-cultures with tumor cells as detected by ELISA. In standard 51Cr cytotoxicity assays, TanCAR T cells distinctly recognized and killed HER2 and GD2 expressing OS cells as well as tumor cells expressing either HER2 or GD2 only. Importantly, bispecific TanCAR T cells attained full activation only upon encounter of both target antigens simultaneously yet they sustained their anti-tumor activity at low T-cell to tumor cell ratios and at modest or absent expression of either. CONCLUSION: The described bispecific single chimeric antigen receptor molecule (TanCAR) could widen the therapeutic application of a T-cell product by recognition of either target antigens (the Boolean OR), offsetting antigen escape. Importantly, if could improve selective full T cell activation (the Boolean AND) to maintain safety by minimizing off-target effects. Citation Format: Meenakshi G. Hegde, Amanda Corder, Tiara T. Byrd, Kristen Fousek, Helen E. Heslop, Stephen Gottschalk, Eric Yvon, Gianpietro Dotti, Nabil Ahmed. A rationally designed bispecific chimeric antigen receptor molecule that simultaneously redirects T cells to target HER2 and GD2 in osteosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-199. doi:10.1158/1538-7445.AM2014-LB-199

Adoptive T-Zell-Therapie des Rhabdomyosarkoms

To improve survival of patients with advanced rhabdomyosarcomas (RMS), we aimed to adoptively transfer T-cells with redirected specificity for the fetal acetylcholine receptor (AChR), an RMS-specific cell surface antigen. A "second generation" chimeric antigen receptor (CAR) with a combined CD28-CD3ζ signaling domain was derived from our previously described chimeric antigen receptor composed of an extracellular human anti-fAChR antibody fragment, an Fc hinge region, and the intracellular T-cell receptor zeta chain. Lymphocytes from the peripheral blood were modified by retroviral transduction and monitored by FACS analysis. Cytotoxicity of modified T-cells towards RMS cells was recorded by MTT-based viability tests; expression of co-stimulatory molecules and anti-apoptotic genes was studied by FACS and qRT-PCR analysis. Co-stimulatory molecules were expressed in low levels on RMS cells giving the rationale to generate a CD28-CD3ζ signalling CAR (chimeric antigen receptor) for redirecting T-cells. T-cells were successfully engineered with the "second generation" AChR-specific chimeric antigen receptor. Despite of high CAR expression engineered T-cells showed low killing efficiency towards RMS compared to redirected killing of CD20+ lymphoma or CEA-expressing adenocarcinoma cell lines when redirected by CD20- and/or CEA-specific CAR. Data suggest that RMS cells exhibit resistance to a T-cell attack redirected by a fAChR-specific CAR. Inhibition of anti-apoptotic pathways in those cells may improve sensitivity to conventional as well as T-cell-based therapeutics.

Packed Red Blood Cells Suppress T-Cell Proliferation Through a Process Involving Cell–Cell Contact

Packed red blood cell (PRBC) transfusion suppresses immunity and increases morbidity and mortality. Leukocyte reduction has failed to abrogate these effects, thus implicating red blood cells themselves or their components. PRBC impair proliferation of immortal (Jurkat) T cells by depleting arginine from the extracellular environment. The effect of PRBC on isolated ex vivo T-cell proliferation has not been reported. We hypothesize that PRBCs depress mitogen-stimulated proliferation in isolated human and mouse T cells. Human peripheral T cells were isolated by Ficoll-Hypaque gradient, purified by magnetic separation, and stimulated with anti-CD3 or anti-CD28. DO11.10 transgenic mouse splenic T cells were stimulated with ovalbumin. Cells were cultured at 1 x 10(6)/mL in 96-well plates or in 24-transwell plates in the presence of PRBC (0.015-5% by volume, stored for 4-6 weeks). In culture media, arginine and citrulline were varied. Proliferation was measured at 72 hours by thymidine incorporation. T-cell viability, apoptosis, and receptor zeta chain were measured by flow cytometry. PRBC significantly depressed human peripheral and mouse splenic T-cell proliferation in a dose-dependent manner. PRBC arginase blockade by N-omega-hydroxy-nor-l-arginine only partly restored proliferation. Cell contact was required in both cell types for maximal effect. Depressed zeta chain in human peripheral T cells was partly restored by arginase blockade. Salvage by high-dose arginine and citrulline was unsuccessful. Decreased proliferation was not related to cell death. PRBC suppresses mitogen-stimulated human and antigen-stimulated mouse T-cell proliferation by mechanisms independent of arginine depletion. This is a novel mechanism for transfusion-associated immune suppression.

Uncoupling of T Cell Receptor Zeta Chain Function during the Induction of Anergy by the Superantigen, Staphylococcal Enterotoxin A

Staphylococcus aureus enterotoxins have immunomodulatory properties. In this study, we show that Staphylococcal enterotoxin A (SEA) induces a strong proliferative response in a murine T cell clone independent of MHC class II bearing cells. SEA stimulation also induces a state of hypo-responsiveness (anergy). We characterized the components of the T cell receptor (TCR) during induction of anergy by SEA. Most interestingly, TCR zeta chain phosphorylation was absent under SEA anergizing conditions, which suggests an uncoupling of zeta chain function. We characterize here a model system for studying anergy in the absence of confounding costimulatory signals.

Host immune responses to cervical cancer

The purpose of this review is to understand the role of the host immune system in clearing the human papillomavirus (HPV) infection, strategies adopted by HPV to subvert the host immune responses and analyze the challenges to the future immunotherapeutic treatment modalities. Cervical epithelium provides a protective niche to the virus to subvert the immune responses. The absence of an inflammatory milieu in the cervix makes the resident dendritic and langerhan cells tolerogenic to HPV antigens. CD4 cells predominated in regressing cervical intraepithelial neoplasia lesions, whereas CD8 cells were dominant in invasive carcinoma. A reduced expression of T cell signaling molecule T-cell receptor zeta chain was observed in CD8 lymphocytes. Decreased numbers of NKG2D expressing natural killer and T cells were present in patients with cervical cancer and cervical intraepithelial neoplasia. Increased frequencies of CD4 CD25+ FoxP3+ T regulatory cells were observed in patients with cervical cancer. The Nrp-1+Treg showed greater suppressive activity. A network of Treg and indoleamine 2, 3-dioxygenase expressed in tumor cells facilitates immune escape of tumor cells. The HPV uses different strategies to evade immune recognition. Understanding the immune evasion mechanisms used by HPV will help in designing newer therapeutic strategies for cervical cancer.

Mechanisms involved in the down‐regulation of TCR ζ chain in tumor versus peripheral blood of oral cancer patients

Immune dysfunction is the hallmark of patients with oral cancer. Down-regulation of T cell receptor (TCR) zeta chain expression was observed in T cells from patients with oral squamous cell carcinoma. In peripheral blood, the decrease in TCR zeta chain showed an inverse correlation with the tumor stage as demonstrated by western blotting, confocal microscopy and flow cytometry. The mechanism of TCR zeta chain degradation in the peripheral blood involves ubiquitination and subsequent targeting of TCR zeta for degradation in the lysosome. Decreased expression of PKC theta and the subsequent decrease of TCR zeta chain transcription factor Elf-1 and its binding to DNA may contribute to the decreased/or absent TCR zeta chain transcripts in the tumor infiltrating lymphocytes. Oral cancer patients exhibiting TCR zeta chain defect also showed impaired lymphocyte proliferation, cytokine profile and intracellular calcium release upon stimulation with anti CD3 mAb. Our data shows that posttranslational degradation is primarily responsible for decreased TCR zeta chain expression in the peripheral blood, while a transcriptional defect is observed in the tumor compartment. The down-regulation of TCR zeta chain culminates into impaired lymphocyte responses in these patients.

The Feature of TCR Zeta Gene in CD4+ and CD8+ T Cells in Patients with CML.

The T-cell receptor (TCR) zeta chain is a master sensor and regulator of lymphocyte responses which plays a critical role in TCR-mediated signal transduction. The abnormal expression of TCR zeta gene was found in some malignancies and immune disease. Cellular immune deficiency is the common feature in patients with chronic myeloid leukemia (CML). Our previous studies had showed that 60% of CML patients displayed an abnormally low expression of TCR zeta chain in peripheral blood mononuclear cells (PBMCs). To further estimate the changes of zeta chain expression of T-lymphocyte subsets in CML, TCR zeta chain gene expression level in purified CD4+ and CD8+ T cells sorted by MACS were detected by real-Time PCR with SYBR Green I technique, CD4+ and CD8+ T cells from both 10 cases with CML and 10 normal individuals were selected to analyze in the study. β2 -microglobulin was used as an endogenous reference. Relative changes in TCR zeta gene expression level were used by the 2−ΔCt×100% method. The relative mRNA expression level of TCR zeta gene in CD4+ and CD8+ T cells from CML was 4.06±4.82% and 3.82±4.25% respectively, whereas 7.75±2.52% and 5.19±1.25% were found in normal individuals. There was a wide range in the TCR zeta gene expression level of CD4+ T cells from CML patients (0.23–15.93%,) with a median value of 1.77%, whereas the a range of (4.74–12.81%) with a median of 7.93% was showed in normal controls (P=0.046). The expression level of TCR zeta gene in CD8+ T cells from controls ranged from 3.47% to 6.75% (median 5.52%) and in CML patients from 0.86% to 15.55% with a median of 2.51% (P=0.340). The results indicated that, compared with normal controls, zeta chain gene expression was obviously down-regulated in CD4+ T cells from patients with CML. However, there was not significant different in expression level of TCR zeta gene of CD8+ T cells between CML patients and normal individuals. Moreover, the expression level of TCR zeta gene is nonsignificant age-associated or gender-link in CD4+ T cells and CD8+ T cells from patients with CML. In conclusions, the results provide at first the difference of TCR-zeta gene expression pattern in CD4+ and CD8+ T cells from patients with CML, this may be reflective of signaling difference between the different T cell subtypes in immunodeficiency state of patients.

Decreased TCR ζ‐chain expression in T cells from patients with acquired aplastic anaemia

In aplastic anaemia, T cells have a central role in the pathophysiology of bone marrow destruction. This study showed that T cells from patients with aplastic anaemia expressed decreased T-cell receptor (TCR) zeta-chain protein and mRNA levels compared to healthy controls. Patients with decreased TCR zeta-chain showed an abnormal response in intracellular calcium following stimulation through the TCR. We also observed an altered pattern of the transcription factors CREMalpha and Elf-1 that are implicated in zeta-chain transcription. We concluded that TCR zeta-chain expression was decreased in the majority of patients with aplastic anaemia, regardless of disease activity or treatment status.

Structure Induction of the T‐Cell Receptor ζ‐Chain upon Lipid Binding Investigated by NMR Spectroscopy

The conformation of the cytoplasmic part of the zeta-chain of the T-cell receptor (TCR) in its free form and bound to detergent micelles has been investigated by heteronuclear NMR spectroscopy. The zeta-chain is considered to be a mediator between the extracellular antigen and the intracellular signal-transduction cascade leading to T-cell activation. Earlier studies suggested a T-cell activation mechanism that involved a TCR-state-dependent lipid incorporation propensity of the zeta-chain accompanied by a helical folding transition. In order to support this proposed mechanism, standard protein NMR assignment and secondary-structure-elucidation techniques have been applied to the free TCR zeta-chain and to the zeta-chain bound to the detergent LMPG, which forms a micelle, in order to obtain the structural characteristics of this folding transition in a residue-resolved manner. We could assign the resonances of the free zeta-chain at 278 K, and this formed the basis for chemical-shift-perturbation studies to identify lipid binding sites. Our NMR results show that the free TCR zeta-chain is indeed intrinsically unstructured. Regions around the ITAM2 and ITAM3 sequences are involved in a highly dynamic binding of the free zeta-chain to a detergent micelle formed by the acidic lipid LMPG.

Different T-cell Receptor (TCR) Zeta Chain Expression in Cervical Cancer and its Precursor Lesions

Cervical cancer is associated with infection of epithelial cells with the human papillomavirus (HPV) type 16 and HPV18. A functional signalling machinery in T-cells is required in order to successfully fight and eradicate HPV16+ transformed epithelial cells. One of the key signalling molecules associated with the T-cell receptor (TCR) is the homodimeric zeta chain molecule. 28 formalin fixed und paraffin embedded samples of cervical tissue with cervical intraepithelial lesions CIN I (n = 3), CIN III (n = 7), invasive cervical carcinoma (CC) (n = 13) and normal cervical tissue (n = 5) has been evaluated for HPV-PCR und zeta chain immunohistochemistry. For immunohistochemistry a monoclonal IgG1 anti TZR zeta chain-antibody (mAb) has been used (clone 6B 10.2, Santa Cruz, Heidelberg, Germany). According to the performed Western-Blot analysis on peripheral blood monocytes (PBMCs) the used mAb has specifically recognized TCR zeta chains. We show reduced protein zeta chain expression associated with invasive cervical cancer, but not with pre-invasive HPV16-positive lesions or HPV16-negative normal cervix tissue. Thus, reduced TCR zeta chain expression is not necessarily linked to a chronic viral infection, nor to the presence of transformed cells, but rather to the stromal invasion of the cancer lesion.

TCRζ mRNA splice variant forms observed in the peripheral blood T cells from systemic lupus erythematosus patients

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease of unknown etiology. Tyrosine phosphorylation and protein expression of the T-cell receptor zeta chain (zeta) have been reported to be significantly decreased in SLE T cells. In addition, zeta mRNA with alternatively spliced 3' untranslated region (zetamRNA/as-3'UTR) is detected predominantly in SLE T cells, and aberrant zeta mRNA accompanied by the mutations in the open reading frame including zeta mRNA lacking exon7 (zetamRNA/exon7-) is observed in SLE T cells. These zeta mRNA splice variant forms exhibit a reduction in the expression of TCR/CD3 complex and zeta protein on their cell surface due to the instability of zeta mRNA splice variant forms as well as the reduction in interleukin (IL)-2 production after stimulating with anti-CD3 antibody. Data from cDNA microarray showed that 36 genes encoding cytokines and chemokines, including IL-2, IL-15, IL-18, and TGF-beta2, were down-regulated in the MA5.8 cells transfected with the zeta mRNA splice variant forms. Another 16 genes were up-regulated and included genes associated with membranous proteins and cell damage granules, including the genes encoding poliovirus-receptor-related 2, syndecan-1, and granzyme A.

TCR zeta-chain downregulation: curtailing an excessive inflammatory immune response.

The T-cell receptor (TCR) functions in both antigen recognition and signal transduction, which are crucial initial steps of antigen-specific immune responses. TCR integrity is vital for the induction of optimal and efficient immune responses, including the routine elimination of invading pathogens and the elimination of modified cells and molecules. Of the TCR subunits, the zeta-chain has a key role in receptor assembly, expression and signalling. Downregulation of TCR zeta-chain expression and impairment of T-cell function have been shown for T cells isolated from hosts with various chronic pathologies, including cancer, and autoimmune and infectious diseases. This review summarizes studies of the various pathologies that show this phenomenon and provides new insights into the mechanism responsible for downregulation of zeta-chain expression, its relevance and its clinical implications.