T-cell Surface Research Articles

Protein-Protein Networks Construction and Their Relevance Measurement Based on Multi-Epitope-Ligand-Kartographie and Gene Ontology Data of T-Cell Surface Proteins for Polymyositis

Polymyositis is an inflammatory myopathy characterized by muscle invasion of T-cells penetrating the basal lamina and displacing the plasma membrane of normal muscle fibers. In order to understand the different adhesive mechanisms at the T-cell surface, Schubert randomly selected 19 proteins expressed at the T-cell surface and studied them using MELK technique [4], among which 15 proteins are picked up for further study by us. Two types of functional similarity networks are constructed for these proteins. The first type is MELK similarity network, which is constructed based on their MELK data by using the McNemar's test [24]. The second type is GO similarity network, which is constructed based on their GO annotation data by using the RSS method to measuring functional similarity. Then the subset surprisology theory is employed to measure the degree of similarity between two networks. Our computing results show that these two types of networks are high related. This conclusion added new values on MELK technique and expanded its applications greatly.

A novel bispecific single-chain antibody for ADAM17 and CD3 induces T-cell-mediated lysis of prostate cancer cells

ADAM17 (A disintegrin and metalloproteinase 17) is a membrane-bound protease that cleaves various cell surface proteins, including cytokines and cytokine receptors. Recently it was shown that ADAM17 is highly expressed on the surface of many cancer cells, whereas normal cells express low levels of ADAM17, implying that ADAM17 is a potential immunotherapeutic target. We have generated a monoclonal antibody against human ADAM17, which recognized the membrane proximal cysteine-rich extension of the ADAM17 protein. Unlike normal cells, tumour cell lines, such as a prostate cancer cell line, pancreatic cancer cell lines, a breast cancer cell line and a non-small lung cancer cell line, expressed ADAM17 on the cell surface. Using the sequence of the antibody we generated an ADAM17-specific scFv (single-chain variable fragment) and fused this to a CD3-specific scFv to generate a bispecific T-cell engager antibody [A300E-BiTE (bispecific T-cell engager antibody)]. Specificity was demonstrated on cells in which ADAM17 was knocked down with a specific shRNA (short hairpin RNA). A300E-BiTE recognized ADAM17 and CD3 on the cell surface of tumour cells and T-cells respectively. In the presence of primary human peripheral blood mononuclear cells or human T-cells the addition of A300E-BiTE led to ADAM17-specific killing of prostate tumour cells indicating a novel strategy for the treatment of cancer.

Positive conversion of negative signaling of CTLA4 potentiates antitumor efficacy of adoptive T-cell therapy in murine tumor models

AbstractCytotoxic T lymphocyte–associated antigen 4 (CTLA4) has been known to be a strong tolerance-inducing inhibitory receptor on T-cell surface. Systemic blocking of CTLA4 function with blocking antibodies has been regarded as an attractive strategy to enhance antitumor immunity. However, this strategy accompanies systemic autoimmune side effects that are sometimes problematic. Therefore, we developed a novel CTLA4 mutant that could be expressed in tumor antigen-specific T cells to enhance antitumor effect without systemic autoimmunity. This mutant, named CTLA4-CD28 chimera, consists of extracellular and transmembrane domains of CTLA4, linked with cytoplasmic CD28 domain. Overexpression of CTLA4-CD28 chimera in T cells delivered stimulatory signals rather than inhibitory signals of CTLA4 and significantly enhanced T-cell reactivity. Although this effect was observed in both CD4 and CD8 T cells, the effect on CD4 T cells was predominant. CTLA4-CD28 chimera gene modification of CD4 T cells significantly enhanced antitumor effect of unmodified CD8 T cells. Nonetheless, the gene modification of CD8 T cells along with CD4 T cells further maximized antitumor effect of T cells in 2 different murine tumor models. Thus, CTLA4-CD28 chimera gene modification of both tumor antigen-specific CD4 and CD8 T cells would be an ideal way of modulating CTLA4 function to enhance tumor-specific T-cell reactivity.

MTORC2 controls T-cell receptor processing and surface expression during thymocyte development (115.18)

Abstract An efficient immune response relies on the presence of T-cells expressing a functional T-cell receptor (TCR). While the mechanisms generating TCR diversity for antigenic recognition are well defined, what controls its surface expression is less known. The mammalian target of rapamycin (mTOR) forms two structurally and functionally distinct protein complexes, mTOR complex 1 (mTORC1) and mTORC2. While mTORCs were found to regulate T lymphocyte activation and lineage commitment, their role in early thymocyte development remains to be defined. Here we found that mTORC2 modulates T-cell ontogeny and activation by controlling the quantity of TCR that reaches the cell surface of developing thymocytes as well as transformed T-cells. Deletion of the mTORC2 component rictor at early stages of T cell ontogeny (DN2 and onward) led to aberrant maturation and increased proteasomal degradation of nascent TCR polypeptides. Consequently, the levels of TCR as well as other receptors, including CD4, CD8 and CD147 were significantly attenuated on the surface of rictor-deficient thymocytes. Since CD147 was also defectively glycosylated and expressed in SIN1-deficient fibroblasts, our findings suggest that mTORC2 is involved in the co/post-translational processing of membrane receptors. Thus, we uncover a novel function for mTORC2 that provides rationale to target this complex in disorders caused by deregulated expression of surface receptors.

Abstract 4888: Decitabine and vorinostat cooperate to sensitize metastatic human colon carcinoma cells to Fas-mediated apoptosis

Abstract The death receptor Fas and its physiological ligand, FasL, which is expressed on the surface of cytotoxic T-cells (CTLs), regulate apoptosis of cancerous cells. While normal colon epithelial cells express high levels of Fas, Fas expression is diminished in colon carcinomas. Metastatic colon cancers acquire an apopotosis-resistant phenotype often due to the loss of Fas expression and function. Previous studies have implicated Fas-mediated apoptosis as an important contributor of tumor regression. Therefore, targeting Fas resistance is of critical importance in treating colon cancer through Fas-based cancer therapy and immunotherapy. In this study, we explore whether epigenetic pharmacological agents can restore Fas expression and re-establish sensitivity to Fas-mediated apoptosis in metastatic human colon carcinoma cells. First we determined that the epigenetic drugs Decitabine, a DNA methyltransferase inhibitor, and Vorinostat, a HDAC inhibitor, each are capable of up-regulating Fas expression. When combined, these two inhibitors cooperate to increase Fas expression to an even greater extent than each agent alone. Pre-treatment with decitabine, vorinostat, or both drugs followed by incubation with FasL, was able to overcome resistance to FasL-induced apoptosis. Analysis of bisulfite-modified genomic DNA, isolated from human metastatic colon carcinoma cells revealed that the Fas promoter is sporadically methylated; therefore sensitization of Fas receptor expression on the cell surface by decitabine was unlikely through direct inhibition of Fas promoter meythlation. Although the Fas promoter was not heavily methylated, the promoters for BNIP3 and Bik, which are downstream of Fas signaling, were methylated. After treatment with these pharmacological epigenetic inhibitors, alone or in combination, protein levels of both BNIP3 and Bik were increased. Our data suggest that combined modalities of chemotherapy and Fas-based immunotherapy may be an effective approach for the suppression of colon cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4888. doi:1538-7445.AM2012-4888

Abstract LB-421: Dual shRNA technique to screen gene-to-gene interaction

Abstract Background: Synthetic lethality screening in human cells is one of the most powerful tools to identify potential therapeutic targets in a genotype specific manner and this screening has been performed in isogenic models of matched cell-line pairs. The procedure of establishing and isolating isogenic cell lines can be arduous. And despite this laborious work, specific target(s) identified in this specific isogenic cell line may not be valid in other similar cells and experimental conditions. Therefore, we have optimized a fast and robust dual shRNA technique to screen gene-to-gene interaction. Methods and Results: To create a matched cell line pair, we utilized a bicistronic vector containing the transmembrane and extracellular domains of the mouse T-cell surface glycoprotein CD8 alpha, co-expressed with a specific shRNA, to model a specific gene deficiency. This retroviral vector was transduced into target cells, and the positively transduced cells were isolated at two days post-infection using MACS separation columns with mouse CD8a-specific microbeads. This yielded greater than 95% purity. At this time point, the decreased expression of specific gene by shRNA was minimal. After recovery from beads purification, the second bicistronic shRNA vector was introduced. This second vector contained a puromycin selectable marker with GFP and an shRNA targeting the proposed synthetic lethal gene. After second transduction, cells were selected under puromycin allowing two doubling times. The purity was 96-98% based on GFP signal measured by FACS at the end of puromycin selection. The dual-gene knockdown by sequentially introduced shRNAs achieved 70-90% depletion of each gene product. This level of knockdown was maintained for up to three weeks without significant loss of purity. After maintaining the cells in culture for several weeks CD8a purity did decrease to 70-80%, but was restored to 98-99% purity after re-applying the separation columns. The second level of purity was maintained even longer than single purification. Conclusion: In summary, we have developed a fast and efficient dual shRNA technique to apply to shRNA experimentation, obviating the need for establishing conventional isogenic cell line models. Due to its fast and simple procedure, this can be simultaneously performed in multiple cell lines. This technique can easily be adapted to screen shRNA libraries for the discovery of unknown synthetic lethal interactions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-421. doi:1538-7445.AM2012-LB-421

Abstract 3482: Validation of novel therapeutic targets for T-cell non-Hodgkin lymphoma

Abstract T-cell malignancies account for 10-15% of all human non-Hodgkin lymphomas (NHL). T-cell lymphomas are incurable, and the development of effective treatment strategies has been hindered by the relatively infrequency of these conditions. T-cell lymphomas occur spontaneously in dogs; they are morphologically similar to human T-cell lymphomas, resemble their biological behavior, and are relatively common (∼35% of all NHL), providing opportunities to improve our understanding of the pathogenesis of these tumors as well as to develop more effective therapies. Tumor-initiating cells (TICs) influence tumor progression and response to therapy in many cancers, so eliminating these cells may be necessary to achieve durable remissions. However, the existence of TICs and their potential significance have not been conclusively established in T-cell NHL. The objective of this study was to evaluate the existence of progenitor-like lymphoma cells (PLCs) in naturally occurring canine T-cell NHL. We identified a reproducible population of tumor-related PLCs in spontaneous T-cell malignancies. The cells expressed the hematopoietic progenitor antigen CD34 and tumor-specific T-cell receptor gene rearrangements. Somewhat unexpectedly, these cells also express B-cell surface proteins (CD20 and CD22), but not the major T-cell surface antigens (CD3 and CD5) that were expressed by the bulk of the tumor cells (BTCs). We have adapted a xenotransplantation assay for primary canine T-cell NHL using NOD/SCID/IL-2Rgammanull mice and genetically modifying the tumor cells to express luciferase as a means monitor tumor growth and distribution by in vivo imaging. This model allows us to expand and serially transplant primary canine T-cell NHL for more detailed studies, including limiting dilution assays and therapeutic targeting using anti-B-cell reagents. In summary, we propose a novel concept in which a small population of tumor cells with “B-cell progenitor” phenotypes may paradoxically contribute to the pathogenesis of T-cell lymphoma, potentially representing a novel therapeutic target. We already use dogs with naturally occurring disease in the pathway for therapy development, allowing us to expand these studies into clinical trials that can then be rapidly translated to benefit human patients (for example, by using approved agents such as anti-CD20 antibodies or CD22-targeted immunotoxins). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3482. doi:1538-7445.AM2012-3482

In vitro-activated tumor-bearing host T cells and the effectiveness of tumor vaccine immunotherapy

BACKGROUND AND OBJECTIVEVaccination during periods of lymphopenia may facilitate immune responses to weak self-antigens and enhance antitumor immunity. The objective of this study was to determine the effectiveness of tumor vaccine immunotherapy combined with immune reconstruction using tumor-bearing host immune cells in lymphopenia, and to investigate the role of tumor-bearing host T cells activated in vitro during immunotherapy.DESIGN AND SETTINGAnimal study conducted in the First Affiliated Hospital of Xi’an Jiaotong University from January 2009 to January 2010.PATIENTS AND METHODSLymphopenia was induced by cyclophosphamide. A reconstituted immune system with different syngeneic lymphocytes was employed, including lymphocytes from naïve rats (unsensitized group), tumor-bearing rats (tumor-bearing group), and tumor-bearing rats activated in vitro (activated group). All rats were immunized with granulocyte-macrophage colony-stimulating factor (GM-CSF)-modified NuTu-19 ovarian cancer (GM-CSF/NuTu-19) cells. Tumor vaccine-draining lymph nodes (TVDLNs) were harvested, and then stimulated to induce effector T cells (TE). TE were then adoptively transferred to rats bearing a 3-day pre-established abdominal tumor (NuTu-19), and the survival rate was calculated.RESULTSCompared with the unsensitized group, the levels of interleukin-2 (IL-2) were significantly lower in the tumor-bearing group, whereas that of IL-4 were significantly higher (P<.05). The number of CD4+ T cells secreting interferon-γ and the specific cytotoxicity of CD8+ cytotoxic T lymphocytes were significantly lower (P<.05). The survival was significantly higher in the activated group compared with the other groups.CONCLUSIONSLymphocytes from tumor-bearing rats activated in vitro can effectively reverse the immunosuppressive effects of tumor-bearing hosts.

Progressive Activation of CD127+132− Recent Thymic Emigrants into Terminally Differentiated CD127−132+ T-Cells in HIV-1 Infection

AimHIV infection is associated with distortion of T-cell homeostasis and the IL-7/IL7R axis. Progressive infection results in loss of CD127+132− and gains in CD127−132+ CD4+ and CD8+ T-cells. We investigated the correlates of loss of CD127 from the T-cell surface to understand mechanisms underlying this homeostatic dysregulation.MethodsPeripheral and cord blood mononuclear cells (PBMCs; CBMC) from healthy volunteers and PBMC from patients with HIV infection were studied. CD127+132−, CD127+132+ and CD127−132+ T-cells were phenotyped by activation, differentiation, proliferation and survival markers. Cellular HIV-DNA content and signal-joint T-cell receptor excision circles (sjTRECs) were measured.ResultsCD127+132− T-cells were enriched for naïve cells while CD127−132+ T-cells were enriched for activated/terminally differentiated T-cells in CD4+ and CD8+ subsets in health and HIV infection. HIV was associated with increased proportions of activated/terminally differentiated CD127−132+ T-cells. In contrast to CD127+132− T-cells, CD127−132+ T-cells were Ki-67+Bcl-2low and contained increased levels of HIV-DNA. Naïve CD127+132− T-cells contained a higher proportion of sjTRECs.ConclusionThe loss of CD127 from the T-cell surface in HIV infection is driven by activation of CD127+132− recent thymic emigrants into CD127−132+ activated/terminally differentiated cells. This process likely results in an irreversible loss of CD127 and permanent distortion of T-cell homeostasis.

Monitoring of Disease Biomarkers Activity and Immunophenotyping as Important Factors in SLE Clinical Management

The highly specific biomarkers for monitoring of SLE disease activity are not yet defined up to date, due to existing of different clinical SLE phenotypes caused by individual genetic variation. Basically, numerous clinical complications follow SLE patients such as nephritis, atherosclerosis and cardial, CNS, gastrointestinal and ophthalmological complications, as well. Their monitoring in clinical SLE management can be evaluated by analysing of specific biochemical parameters and require permanent clinical observation. The presence of ANAs and anti-ds-DNAs are usual diagnostic SLE autoimmunity parameters, while SLE disease activity biomarkers are C3 and C4 level, anticardiolipin antibodies, anti-Sm/RNPs and, recently level of CD4 and CD8 lymphocytes. However, the number of TCR molecules on the T-cells surface at SLE patients is lower then in normal condition, and otherwise for these receptors CD molecules make specific connection. On the other hand, the T lymphocytes can be also, therapeutical targets at SLE patients, because of their clear direct involving in SLE pathogenesis. The SLE phenotypes are characterized by double CD negativity ( CD3 +/-, CD4-) caused by abnormal level of IL-2 and IL-17. T-lymphocytes have usually alpha-beta and gamma-delta TCR receptors, but for SLE patients is characteristic lower number gama-delta TCR molecules, detected in the peripheral blood specimens. Taking into account all of the facts, we investigated the level of specific usual SLE activity biomarkers (anti-ds-DNAs, C3, C4, anticardiolipin antibodies (beta-2-IgG, beta-2-IgM, ACA-G, ACA-M, CD4 and CD8 level) in serum specimens of SLE patients who underwent to the corresponding chemotherapy in combination with other biochemical and clinical parameters. Once again proved to be, that SLE biomarker monitoring, could be useful aproach for SLE activity disease and prediction organ damage, as well. In our investigation we used the following methods: immunofluorescence microscopy (IFA-ANA), and nephelometry, Hycor ELISA system and Flow cytometry, for precisely quantitative measurements. We determined correlation between C3 and C4 complement components level, CD3 (T-Ly), CD3+/HLA-DR and total HLA-DR with regard to SLE disease activity. Also, CD4 (Th), CD4:CD8 ratio, beta-2-G, beta-2-M not proved to be useful biomarkers in this sense, despite some results specific for some special SLE phenotypes. Anti-Sm/ RNPs proved to be better in SLE diagnostic process.

Associations of killer cell immunoglobulin- like receptor genes with rheumatoid arthritis.

Objective: Rheumatoid Arthritis (RA) is an autoimmune and chronic inflammatory disease of unknown etiology. Killer cell immunoglobulin-like receptors are expressed on the surface of natural killer cells and CD28null T-cells, both present in synovial membrane of RA. Therefore we evaluated the associations of KIR genes with RA. Methods: 16 KIR genes were genotyped in 100 healthy subjects (HS) and 100 RA patients from Western Mexico using PCR-SSP. Differences in KIR genotypes and gene frequencies were assessed using the X2 test. Results: Gene frequency of KIR2DL3 was lower in RA than in HS (p = 0.0019), whereas KIR2DL2 and KIR2DS2 were higher in RA than HS (p = 0.0004 and p = 0.0487, respectively). In addition were identified 38 genotypes (from G1-G38) in both studied groups, and the genotype frequencies of G1, G6 and G14 showed significant differences (p = 0.0001, p = 0.0208 and p = 0.0300, respectively). Conclusions: The presence of KIR2DL2, KIR2DS2 and absence of KIR2DL3 are associated with RA. Moreover, two genotypes BX are associated with RA. These results suggest that KIRs can be involved in RA susceptibility.

Intravenous Administration of Glutamate Ionotropic Antagonists in Healthy Rats, Induces Lymphopenia and Affects Their Hematological Profile

Background: Glutamate is one of the primary endogenous amino acids of the Central Nervous System (CNS). Research over the last years has proven the existence of glutamate subunit receptors in additional non-neuronal tissues outside CNS such as the hematopoietic system.The purpose of this paper is to define the possible in vivo effect of glutamate ionotropic antagonists MK-801and NBQX in the hematopoietic system as well as their possible use in the cure of hematopoietic diseases and on their use on patients with co-existing neurological and hematopoietic disorders. Methods : Glutamate ionotropic antagonists MK-801 and NBQX were administrated intravenously in male wistar rats of 250 - 350 g weight. Animals treated with glutamate antagonists (n = 24) were compared to a control group (n = 10). The following parameters were evaluated 12 h and 24 h after drug administraton: Full blood count with differential, aggregation intensity, plus CD3, CD54 and CD49a expression on T-lymphocytes with flow cytometry. Bone marrow was assessed with bone marrow smears. The results were analyzed by unpaired t-test. Results : Both ionotropic antagonists reduced white blood cells with early lymphopenia (P less than 0.05), whereas NBQX also increased Ht and Hb values (P < 0.05), but reduced platelet aggregation (P less than 0.05). T-cell surface activation markers, CD3, CD49a were reduced (P less than 0.05), whereas CD54 was increased (P less than 0.05) 12 h after both antagonists’ administration but 24 h after NBQX all markers were increased (P less than 0.05). Bone marrow cells were influenced in a way that peripheral blood changes and segmented cells showed a possible expression of glutamate receptors. Conclusions : It is concluded that glutamate ionotropic antagonists affect the hematological profile and bone marrow of healthy intact rats as well as blood elements functions. Further research is needed in order to define the in vivo effect of glutamate and glutamate receptor antagonists on hematopoietic system either as a toxic agent or as a therapeutical one. doi:10.4021/jh101e

Immune-Regulatory Mechanisms in Systemic Autoimmune and Rheumatic Diseases

Immune-Regulatory Mechanisms in Systemic Autoimmune and Rheumatic Diseases

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Raltegravir Treatment Intensification Does Not Alter Cerebrospinal Fluid HIV-1 Infection or Immunoactivation in Subjects on Suppressive Therapy

Despite suppression of plasma human immunodeficiency virus type 1 (HIV-1) RNA by antiretroviral therapy to levels below clinical assay detection, infection and immune activation may persist within the central nervous system and possibly lead to continued brain injury. We hypothesized that intensifying therapy would decrease cerebrospinal fluid (CSF) infection and immune activation. This was a 12-week, randomized, open-label pilot study comparing addition of the integrase inhibitor raltegravir to no treatment augmentation, with an option for rollover to raltegravir. CSF and plasma were analyzed for HIV-1 RNA using a single-copy assay. CSF and blood immune activation was assessed by neopterin concentrations and CD4(+) and CD8(+) T-cell surface antigen expression. Primary analysis compared 14 intensified (including rollovers) to 9 nonintensified subject experiences. Median HIV-1 RNA levels in all samples were lower in CSF (<.3 copies/mL) than in plasma (<.9 copies/mL; P < .0001), and raltegravir did not reduce HIV-1 RNA, CSF neopterin, or CD4(+) and CD8(+) T-cell activation. Raltegravir intensification did not reduce intrathecal immunoactivation or alter CSF HIV-1 RNA levels in subjects with baseline viral suppression. With and without raltegravir intensification, HIV RNA levels in CSF were very low in the enrolled subjects. Clinical Trials Registration. NCT00672932.

Ursolic acid inhibits T-cell activation through modulating nuclear factor-κ B signaling

To investigate the effects of ursolic acid (UA) on T-cell proliferation and activation, as well as to examine its effect on nuclear factor-κB (NF-κB) signaling pathway in T cells. T-cells isolated from BALB/c mice were incubated with UA at concentrations ranging from 5-30 μmol/L in the presence of phorbol 12-myristate 13-acetate (PMA) or PMA plus ionomycin. The proliferation of T cells was measured by the MTT assay. The expressions of CD69, CD25, and CD71 on T-cell surface were analyzed using flow cytometry. The level of interleukin-2 (IL-2) in the culture supernatant of activated T cells was quantified by enzyme-linked immunosorbent assay (ELISA). The level of phosphorylated IκB-α (p-IκB-α) in total protein and p65, a subunit of NF-κB, nuclear translocation were measured by Western blot analysis. UA in a dose-dependent manner significantly decreased the proliferation and inhibited the surface expressions of CD69, CD25, and CD71 in murine T lymphocytes upon in vitro activation (P<0.01). Significant reduction of IL-2 production was found in activated T cells treated with UA (P<0.01). The PMA-induced increase in p-IκB-α protein was inhibited, and nuclear translocation of p65 from the cytoplasm was blocked by UA. UA is a potent inhibitor for T cell activation and proliferation; these effects are associated with the inhibition of NF-κB signaling pathway.

Cell Surface Delivery of TRAIL Strongly Augments the Tumoricidal Activity of T Cells

Adoptive T-cell therapy generally fails to induce meaningful anticancer responses in patients with solid tumors. Here, we present a novel strategy designed to selectively enhance the tumoricidal activity of T cells by targeted delivery of TNF-related apoptosis-inducing ligand (TRAIL) to the T-cell surface. We constructed two recombinant fusion proteins, anti-CD3:TRAIL and K12:TRAIL. Tumoricidal activity of T cells in the presence of these fusion proteins was assessed in solid tumor cell lines, primary patient-derived malignant cells, and in a murine xenograft model. When added to T cells, K12:TRAIL and anti-CD3:TRAIL selectively bind to the T-cell surface antigens CD3 and CD7, respectively, leading to cell surface accretion of TRAIL. Subsequently, anti-CD3:TRAIL and K12:TRAIL increased the tumoricidal activity of T cells toward cancer cell lines and primary patient-derived malignant cells by more than 500-fold. Furthermore, T-cell surface delivery of TRAIL strongly inhibited tumor growth and increased survival time of xenografted mice more than 6-fold. Targeted delivery of TRAIL to cell surface antigens of T cells potently enhances the tumoricidal activity of T cells. This approach may be generally applicable to enhance the efficacy of adoptive T-cell therapy.

Analysis of Differentially Expressed Proteins in Colorectal Cancer Using Hydroxyapatite Column and SDS-PAGE

Limitation on two dimensional (2D) gel electrophoresis technique causes some proteins to be under presented, especially the extreme acidic, basic, or membrane proteins. To overcome the limitation of 2D electrophoresis, an analysis method was developed for identification of differentially expressed proteins in normal and cancerous colonic tissues using self-pack hydroxyapatite (HA) column. Normal and cancerous colon tissues were homogenized and proteins were extracted using sodium phosphate buffer at pH 6.8. Protein concentration was determined and the proteins were loaded unto the HA column. HA column reduced the complexity of proteins mixture by fractionating the proteins according to their ionic strength. Further protein separation was accomplished by a simple and cost effective sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. The protein bands were subjected to in-gel digestion and protein analysis was performed using electrospray ionization (ESI) ion trap mass spectrometer. There were 17 upregulated proteins and seven downregulated proteins detected with significant differential expression. Some of these proteins were low abundant proteins or proteins with extreme pH that were usually under presented in 2D gel analysis. We have identified brain mitochondrial carrier protein 1, T-cell surface glycoprotein CD1a, SOSS complex subunit B2, and Protein Jade 1 which were previously not detected in 2D gel analysis method.

Current Concepts of Hyperinflammation in Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) is the most common inherited disorder of phagocytic functions, caused by genetic defects in the leukocyte nicotinamide dinucleotide phosphate (NADPH) oxidase. Consequently, CGD phagocytes are impaired in destroying phagocytosed microorganisms, rendering the patients susceptible to bacterial and fungal infections. Besides this immunodeficiency, CGD patients suffer from various autoinflammatory symptoms, such as granuloma formation in the skin or urinary tract and Crohn-like colitis. Owing to improved antimicrobial treatment strategies, the majority of CGD patients reaches adulthood, yet the autoinflammatory manifestations become more prominent by lack of causative treatment options. The underlying pathomechanisms driving hyperinflammatory reactions in CGD are poorly understood, but recent studies implicate reduced neutrophil apoptosis and efferocytosis, dysbalanced innate immune receptors, altered T-cell surface redox levels, induction of Th17 cells, the enzyme indolamine-2,3-dioxygenase (IDO), impaired Nrf2 activity, and inflammasome activation. Here we discuss immunological mechanisms of hyperinflammation and their potential therapeutic implications in CGD.

Crystallization and preliminary X-ray crystallographic studies of swine CD8α

CD8αα homodimers or CD8αβ heterodimers form on the T-cell surface, where they are essential as co-receptors for MHC class I molecules in activation of the CTL response. To date, swine have been found to show the highest percentage of lymphocytes with surface expression of CD8α. Crystallographic analysis of swine CD8α (sCD8α) to 1.8 Å resolution revealed that the crystals belonged to space group P3(2)21, with unit-cell parameters a = 80.97, b = 80.97, c = 95.19 Å. The Matthews coefficient and the solvent content were calculated to be 3.23 Å(3) Da(-1) and 61.89%, respectively. These results may aid further structural and functional analyses of sCD8α.