Generation of human neutralizing monoclonal antibodies against the 2009 pandemic H1N1 virus from peripheral blood memory B lymphocytes.

The 2009 H1N1 influenza pandemic demonstrated the significance of a global health threat to human beings. Although pandemic H1N1 vaccines have been rapidly developed, passive serotherapy may offer superior immediate protection against infections in children, the elderly and immune-compromised patients during an influenza pandemic. Here, we applied a novel strategy based on Epstein-Barr virus (EBV)-immortalized peripheral blood memory B cells to screen high viral neutralizing monoclonal antibodies (MAbs) from individuals vaccinated with the 2009 pandemic H1N1 vaccine PANFLU.1. Through a massive screen of 13 090 immortalized memory B-cell clones from three selected vaccinees, seven MAbs were identified with both high viral neutralizing capacities and hemagglutination inhibition (HAI) activities against the 2009 pandemic H1N1 viruses. These MAbs may have important clinical implications for passive serotherapy treatments of infected patients with severe respiratory syndrome, especially children, the elderly and immunodeficient individuals. Our successful strategy for generating high-affinity MAbs from EBV-immortalized peripheral blood memory B cells may also be applicable to other infectious or autoimmune diseases.

Medical education: changes and perspectives.

As medical education undergoes significant internationalization, it is important for the medical education community to understand how different countries structure and provide medical education. This article highlights the current landscape of medical education in China, particularly the changes that have taken place in recent years. It also examines policies and offers suggestions about future strategies for medical education in China. Although many of these changes reflect international trends, Chinese medical education has seen unique transformations that reflect its particular culture and history.

Potential regulatory role of in vitro-expanded Vδ1 T cells from human peripheral blood.

γδ T cells represent a relevant proportion of T lymphocytes that express T cell receptors (TCRs) encoded by the γ and δ T cell receptor genes. Whereas the most circulating γδ T cell type, Vδ2 T cells, has been described and studied intensively, the potential role of Vδ1 T cells remains largely unclear. Here, we identified that expanded peripheral blood Vδ1 T cells stimulated with anti-human TCR Vδ1 monoclonal antibody (mAb) in vitro predominantly expressed forkhead box p3 (Foxp3). In addition, these cells also expressed other regulatory T cell-related molecules, such as CD25, glucocorticoid-induced TNFR family-related protein and cytotoxic T lymphocyte-associated protein-4 (CTLA-4), and held the potent capacity for the production of transforming growth factor beta 1 (TGF-ß1). These autocrine and/or paracrine TGF-ß1 could bind TGF-ß1 receptors on Vδ1 T cells and induce sustained Foxp3 expression. Moreover, Foxp3 expression coincided with high CD25 expression. CD25(high) Vδ1 T cells exhibited stronger suppression on CD4(+) T cell proliferation compared with TGF-ß1-induced CD25(high) CD4(+) regulatory T cells. Therefore, our phenotypic and functional analyses first demonstrate the potential regulatory property of anti-human TCR Vδ1 mAb-activated Vδ1 T cells. These results will broaden our understanding about the role of peripheral blood Vδ1 T cells under physical and pathological conditions.

Aberrant CD40-induced NF-κB activation in human lupus B lymphocytes.

Auto-reactive B lymphocytes and its abnormal CD40 signaling play important roles in the pathogenesis of systemic lupus erythematosus (SLE). In this study, we analyzed CD40 expression and CD40/CD154 induced activation of NF-κB signaling pathway in B cells from SLE patients. B cells from healthy volunteers and tonsilar B cells from chronic tonsillitis were used as negative and positive controls. Results showed CD40-induced NF-κB signaling was constitutively activated in B cells from active lupus patients, including decreased CD40 in raft portion, increased phosphorylation and degradation of IκBα, phosphorylation of P65, as well as increased nuclear translocation of P65, P50, c-Rel, which could be blocked by anti-CD154. CD154 stimulation could induce further phosphorylation and degradation of IκBα, as well as phosphorylation of P65 and nuclear translocation of P65. In addition, CD40-induced kinase activities in B cells from lupus patients mimicked that of tonsil B cells, in that IKKα/ß were more activated compared to normal B cells. CD40-induced NF-κB activity was blocked by both IκB phosphorylation and proteosome degradation inhibitors in both lupus and normal B cells. All together, our findings revealed that canonical NF-κB signaling is constitutively activated in active lupus and is mediated by CD154/CD40. CD40 induced NF-κB activation is different in human lupus B lymphocytes compared with normal B cells.

Aberrant CD200/CD200R1 expression and function in systemic lupus erythematosus contributes to abnormal T-cell responsiveness and dendritic cell activity.

INTRODUCTION: CD200 is a type I transmembrane glycoprotein that can regulate the activation threshold of inflammatory immune responses, polarize cytokine production, and maintain immune homeostasis. We therefore evaluated the functional status of CD200/CD200 receptor 1 (CD200R1) interactions in subjects with systemic lupus erythematosus (SLE). METHODS: Serum CD200 level was detected by ELISA. The expression of CD200/CD200R1 by CD4+ T cells and dendritic cells (DCs) was examined by flow cytometry, and then compared between SLE patients and healthy controls. Peripheral blood mononuclear cells were stained with carboxyfluorescein diacetate succinimidyl ester and annexin V/propidium iodide for evaluation of the effect of CD200 on cell proliferation and apoptosis. In addition, the effect of CD200 on DC function was determined by transwell migration assay as well as by measurement of binding and phagocytosis of apoptotic cells. RESULTS: In SLE patients, the number of CD200+ cells and the level of soluble CD200 were significantly higher than in healthy controls, whereas the expression of CD200R1 by CD4+ T cells and DCs was decreased. Furthermore, the increased CD200 expression by early apoptotic cells contributed to their diminished binding and phagocytosis by DCs in SLE. Importantly, the engagement of CD200 receptor on CD4+ T cells with CD200-Fc fusion protein in vitro reduced the differentiation of T-helper type 17 cells and reversed the defective induction of CD4+CD25highFoxP3+ T cells by transforming growth factor beta in SLE patients. Conversely, blockade of CD200-CD200R1 interaction with anti-CD200R1 antibody promoted CD4+ T-cell proliferation. CONCLUSION: CD200 and CD200R1 expression and function are abnormal in SLE and may contribute to the immunologic abnormalities in SLE.

Abnormal expression of CD43 in patients with systemic lupus erythematosus and its clinical significance.

BACKGROUND: Previous studies indicate that CD43 plays a role in regulating the adhesion of lymphocytes, cell mutation and activation, however, little is known about its effect on systemic lupus erythematosus (SLE). This study was designed to explore the clinical significance of CD43 in SLE patients. METHODS: We used microarray and real-time PCR to detect the mRNA and protein expression of magnetic bead sorted T cells and B cells from peripheral blood mononuclear cells (PBMCs) of SLE patients, and analyzed the relationship between CD43 and the clinical indexes. RESULTS: Both microarray and real-time PCR results showed that CD43 mRNA was significantly decreased in PBMCs of SLE patients compared with healthy controls (P < 0.001). There were no significant differences between lupus nephritis and non-lupus nephritis patients, and neuropsychiatric and non-neuropsychiatric patients. CD43 mRNA expression was significantly reduced in T cells but not in B-cells in SLE patients compared to healthy controls (P < 0.01). Compared with healthy controls, the percentage of CD43(+) cells in the PBMCs of SLE was significantly decreased (P = 0.004), and the CD43 fluorescence intensity in CD3(+)/CD43(+) cells and CD19(+)/CD43(+) cells was also significantly weaker than in healthy controls (P = 0.039 and 0.003). There was no significant difference in the percentage of CD3(+)/CD43(+) cells, CD19(+)/CD43(+) cells between the two groups. The CD43 fluorescence intensity in CD3(+)/CD43(+) cells was inversely correlated with the levels of IgG and IgM (r = -0.8 and -0.6). CONCLUSIONS: Compared to healthy controls, both CD43 mRNA and protein expressions were reduced in T cells from patients with SLE, and were inversely correlated with IgG.

Anti-γδ TCR antibody-expanded γδ T cells: a better choice for the adoptive immunotherapy of lymphoid malignancies.

Cell-based immunotherapy for lymphoid malignancies has gained increasing attention as patients develop resistance to conventional treatments. γδ T cells, which have major histocompatibility complex (MHC)-unrestricted lytic activity, have become a promising candidate population for adoptive cell transfer therapy. We previously established a stable condition for expanding γδ T cells by using anti-γδ T-cell receptor (TCR) antibody. In this study, we found that adoptive transfer of the expanded γδ T cells to Daudi lymphoma-bearing nude mice significantly prolonged the survival time of the mice and improved their living status. We further investigated the characteristics of these antibody-expanded γδ T cells compared to the more commonly used phosphoantigen-expanded γδ T cells and evaluated the feasibility of employing them in the treatment of lymphoid malignancies. Slow but sustained proliferation of human peripheral blood γδ T cells was observed upon stimulation with anti-γδ TCR antibody. Compared to phosphoantigen-stimulated γδ T cells, the antibody-expanded cells manifested similar functional phenotypes and cytotoxic activity towards lymphoma cell lines. It is noteworthy that the anti-γδ TCR antibody could expand both the Vδ1 and Vδ2 subsets of γδ T cells. The in vitro-expanded Vδ1 T cells displayed comparable tumour cell-killing activity to Vδ2 T cells. Importantly, owing to higher C-C chemokine receptor 4 (CCR4) and CCR8 expression, the Vδ1 T cells were more prone to infiltrate CCL17- or CCL22-expressing lymphomas than the Vδ2 T cells. Characterizing the peripheral blood γδ T cells from lymphoma patients further confirmed that the anti-γδ TCR antibody-expanded γδ T cells could be a more efficacious choice for the treatment of lymphoid malignancies than phosphoantigen-expanded γδ T cells.

Generation of human regulatory gammadelta T cells by TCRgammadelta stimulation in the presence of TGF-beta and their involvement in the pathogenesis of systemic lupus erythematosus.

As a component of the innate immune cell population, γδ T cells are involved in tumor immunosurveillance and host defense against viral invasion. In this study, we demonstrated a novel function of human γδ T cells as regulatory cells by detecting their suppressive effect on the proliferation of autologous naive CD4(+) T cells. These regulatory γδ T cells (γδ Tregs) could be generated in vitro by stimulating with anti-TCRγδ in the presence of TGF-ß and IL-2. Similar to CD4(+)Foxp3(+) Tregs, γδ Tregs also expressed Foxp3. Additionally, they primarily belonged to the Vδ1 subset with a CD27(+)CD25(high) phenotype. Furthermore, these γδ Tregs showed an immunoregulatory activity mainly through cell-to-cell contact. Importantly, this γδ regulatory population decreased in the peripheral blood of systemic lupus erythematosus patients, suggesting a potential mechanism in understanding the pathogenesis of systemic lupus erythematosus.

p53 in fibroblast-like synoviocytes can regulate T helper cell functions in patients with active rheumatoid arthritis.

BACKGROUND: p53 is a tumor suppressor and plays a key role in regulating cell hyperplasia, repairing DNA and inducing apoptosis. This study was to investigate p53 expression in fibroblast-like synoviocytes (FLS) and its effect on CD4(+) T lymphocytes from patients with active rheumatoid arthritis (RA). METHODS: Human FLS were transfected with p53 siRNA and cocultured with CD4(+) T lymphocytes from patients with active RA. The expressions of osteoprotegerin and interleukin (IL)-6 were detected in p53 siRNA and scramble siRNA-transfected FLS. In addition, protein levels of interferon (IFN)-γ, IL-17, IL-4 and CD25 as well as mRNAs of IFN-γ, retinoic acid-related orphan receptor (ROR)-γt, IL-17 and Foxp3 in cocultured CD4(+) T lymphocytes were also measured. RESULTS: IL-6 decreased in p53-knockdown FLS while osteoprotegerin expression was not altered. FLS with p53 deletion significantly increased the production of IL-17 and IFN-γ by CD4(+) T cells and upregulated Foxp3 mRNA expression without effects on the proportion of CD4(+)CD25(high) T lymphocytes. CONCLUSION: p53 in FLS might regulate Th1 and Th17 functions in patients with RA and participate in the pathogenesis of RA.

Identify biomarkers of neuropsychiatric systemic lupus erythematosus by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry combined with weak cation magnetic beads.

OBJECTIVE: To identify proteomic biomarkers in cerebrospinal fluid (CSF) and develop a diagnostic proteomic model for neuropsychiatric systemic lupus erythematosus (NPSLE). METHODS: CSF proteomic spectra were generated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) combined with weak cation exchange (WCX) magnetic beads. The spectra were taken from 27 patients with NPSLE before and after treatment, and 27 controls including 17 patients with scoliosis and 10 patients with SLE but without neuropsychiatric manifestation. Discriminating peaks were processed by Biomarker Patterns Software to build a decision tree model for NPSLE classification. In addition, CSF samples of 12 patients with NPSLE, 12 patients with lumbar disc herniation, and 9 patients with other neurological conditions were used as a blind test group to verify the accuracy of the model. RESULTS: Twelve discriminating mass-to-charge (m/z) peaks were identified between NPSLE and controls: m/z peaks 7740, 11962, 8065, 7661, 6637, 5978, 11384, 11744, 8595, 10848, 7170, and 5806. The diagnostic decision tree model, built with a panel of m/z peaks 8595, 7170, 7661, 7740, and 5806, recognized NPSLE with both sensitivity and specificity of 92.6%, based on training group samples, and sensitivity and specificity of 91.7% and 85.7%, respectively, based on the blind test group. In addition, the root node m/z peak 8595 protein, which was downregulated in the CSF of patients with NPSLE after treatment, was identified and confirmed as ubiquitin by immunoprecipitation and ELISA. CONCLUSION: Potential CSF biomarkers for NPSLE are identified by MALDI-TOF-MS combined with WCX magnetic beads. The novel diagnostic proteomic model with m/z peaks 8595, 7170, 7661, 7740, and 5806 is highly sensitive and relatively specific for NPSLE diagnosis. The level of ubiquitin in CSF is a promising biomarker for active NPSLE.

Antigen specificity of gammadelta T cells depends primarily on the flanking sequences of CDR3delta.

The structural basis that determines the specificity of gammadelta T cell receptor (TCR) recognition remains undefined. Our previous data show that the complementary determining region of human TCRdelta (CDR3delta) is critical to ligand binding. Here we used linear and configurational approaches to examine the roles of V, N-D-N, or J regions in CDR3delta-mediated antigen recognition. Surprisingly, we found that the binding activities of CDR3delta from different gammadelta TCRs to their target tissues and ligands depend on the conserved flanking sequences (V and J) but not as much on the D region of CDR3delta fragment. We further defined the key residues in the V and J regions of CDR3delta fragments, including the cysteine residue in the V fragment and the leucine residue in the J fragment that determine their ligand binding specificity. Our results demonstrate that TCRdelta primarily uses conserved flanking regions to bind ligands. This finding may provide an explanation for the limited number of gammadelta TCR ligands that have as yet been identified.

Identification and characterization of Foxp3(+) gammadelta T cells in mouse and human.

Regulatory T cells (Tregs) expressing TCRalphabeta play a critical role in the maintenance of the immune system homeostasis. Tregs express the cell surface markers CD4 and CD25 as well as the transcription factor Foxp3. Foxp3(+)CD4(+)CD25(+)TCRalphabeta(+) Tregs can be generated from mouse and human CD4(+)CD25(-) T cells in vitro via TGF-beta induction. As growing evidences suggest that gammadelta T cells also have immunoregulatory function, we have attempted to identify and characterize Foxp3(+) cells in mouse and human gammadelta T cells. We found that freshly isolated mouse splenic gammadelta T cells did not express Foxp3. When mouse splenocytes were stimulated with anti-TCRgammadelta in the presence of TGF-beta, a population of Foxp3(+) gammadelta T cells appeared, in most of which expressed CD25 as well. Compared with CD25(-) gammadelta T cells, TGF-beta induced CD25(+) gammadelta T cells not only expressed Foxp3, but also had increased TGF-beta and GITR expression. Furthermore, the TGF-beta induced gammadelta T cells mediated a potent immunosuppressive effect on anti-CD3 stimulated T cell activation and proliferation. In contrast, although a small fraction of human peripheral blood and tumor infiltrating gammadelta T cells expressed Foxp3, similar culture condition with anti-TCRgammadelta plus TGF-beta failed to generate functional human Foxp3(+) gammadelta T cells. In conclusion, our results suggest that mouse splenic Foxp3(+) gammadelta T cells with suppressive function can be induced by TCR and TGF-beta costimulation, whereas functional human Foxp3(+) gammadelta T cells in peripheral blood could not be generated under the same condition.

T351 protein, a novel tumor associated antigen and potential target for immunotherapy.

Tumor immunotherapy has attracted many attentions in recent years because of its high specificity. However, the use of immunotherapy is hampered by the lack of defined target tumor antigens. T351 gene, a novel tumor associated gene identified by mRNA differential display, encodes a transmembrane protein. We found that T351 is overexpressed in tumor tissues and tumor cell lines at both mRNA and protein levels by quantitative reverse transcription-PCR, immunohistochemistry and flow cytometry analyses, respectively. We also show that the extracellular portion of T351 protein termed T351pro.philic specifically activate CTLs to lyse tumor cell targets. In addition, a monoclonal antibody raised against T351pro.philic (McAb C1-1) induced murine macrophages to kill tumor cells effectively both in vitro and in vivo. These results suggest that T351 protein is a novel tumor associated antigen.

Identification of human T cell receptor gammadelta-recognized epitopes/proteins via CDR3delta peptide-based immunobiochemical strategy.

Human T lymphocytes, bearing T cell receptor (TCR) gammadelta, play an important role in anti-tumor/microbe immune responses. However, few tumor antigens recognized by TCRgammadelta have been defined so far. To investigate antigenic epitopes/proteins recognized by gammadeltaT cells, we have established a new immunobiochemical strategy that uses complementarity-determining region 3 of TCR delta chain (CDR3delta) peptide-mediated epitope/protein-binding assays. CDR3delta peptides synthesized using the CDR3 region in TCR Vdelta2 chain were validated for their binding specificity to target cells or tissues. These CDR3delta peptides were then employed as probes to pan putative epitopes in a 12-mer random peptide phage-displayed library and to identify putative protein ligands within tumor protein extracts by affinity chromatography and liquid chromatography/electrospray ionization-tandem mass spectrometry analysis. As a result, we have identified nine peptides and two proteins for TCRgammadelta, including human mutS homolog 2 (hMSH2) and heat shock protein (HSP) 60. All nine tested epitope peptides not only bind to gammadeltaT cells but also functionally activate gammadeltaT cells in vitro. Identification of HSP60 confirms the validity of this method as HSP60 is an identified ligand for TCRgammadelta. We show that hMSH2 is expressed on the surface of SKOV3 tumor cells, and cytotoxicity of Vdelta2 gammadeltaT cells to SKOV3 cells was blocked by anti-hMSH2 antibody, suggesting that hMSH2 may be a new ligand for TCRgammadelta. Taken together, our findings provide a novel immunobiochemical strategy to identify epitopes/proteins recognized by gammadeltaT cells.

RAET1E2, a soluble isoform of the UL16-binding protein RAET1E produced by tumor cells, inhibits NKG2D-mediated NK cytotoxicity.

UL16-binding proteins (ULBPs, also termed as retinoic acid early transcripts, encoded by RAET1 genes), a family of ligands for NKG2D in humans, are frequently expressed by tumor cells and mediate cytotoxicities of natural killer (NK) cells and CD8(+) alphabeta T cells to tumor cells. ULBP1, ULBP2, ULBP3, and RAET1L link to membrane through glycosylphosphatidylinositol, whereas RAET1E and RAET1G contain transmembrane and cytoplasmic domains. Proteolytic cleavage of ULBP2 produces truncated and soluble forms that may counteract NKG2D-mediated tumor immune surveillance. In this study, we report that RAET1E can produce a soluble, 35-kDa protein (termed as RAET1E2) lacking the transmembrane region by selective splicing in tumor cells. The expressions of both RAET1E2 transcripts and protein can be found in different tumor cells and tissues. Preincubation of NK-92 cells, a human NK cell line, with culture supernatants from tumor cell lines expressing RAET1E2 or RAET1E2 gene-transfected COS-7 cells resulted in decreased expression of NKG2D on NK-92 cells. Furthermore, incubation of NK-92 cells with recombinant RAET1E2 protein also decreased the surface expression of NKG2D and resulted in marked reduction in cytotoxicities to MGC-803, HepG2, or K562 tumor cells. Taken together, our data provide strong evidence for an immune escape mechanism of tumors via alternative splicing of ULBP RNA to generate a free soluble ULBP protein, RAET1E2, that may impair NKG2D-mediated NK cell cytotoxicity to tumors.

Gammadelta T cells recognize tumor cells via CDR3delta region.

The principles governing gammadelta T cell specificity and diversity remain unclear due to lack of detailed structural analysis. To elucidate key structural basis of the specificity of gammadelta TCR for tumors, we analyzed the binding activities of synthesized TCR Vdelta2 CDR3 peptides derived from tumor infiltrating lymphocyte (TIL) s in ovarian epithelial carcinoma (OEC) via biospecific interaction analysis approach, enzyme immunoassay and immunofluorescence assays. Besides, we used human CDR3delta grafted-Ig to repeat major tests. We found that synthesized OEC-derived CDR3delta peptides could bind specifically to tumor cell lines and tissues. CDR3delta-graft Ig showed a similar binding specificity with CDR3delta peptides, suggesting the determinant role of CDR3delta in antigen binding. Moreover, CDR3delta peptide-mediated binding specificity was blocked by pre-incubation with same peptide, which decreased the cytotoxicity of gammadelta T cells to OEC cells in vitro. Our finding indicates that CDR3delta peptide could mimic antigen-binding specificity of gammadelta TCR. Our strategy provides a novel, simple and convenient approach to investigate the binding activity and function of gammadelta TCR.

A novel recombinant adeno-associated virus vaccine reduces behavioral impairment and beta-amyloid plaques in a mouse model of Alzheimer's disease.

Memory impairment progressing to dementia is the main clinical symptom of Alzheimer's disease (AD). Deposition of the amyloid-beta peptide (Abeta) in brain, particularly its 42-amino acid isoform (Abeta42), has been shown to play a primary and crucial role in the pathogenesis of AD. In this study we have developed a recombinant adeno-associated virus (AAV) vaccine against AD. This vaccine could express CB-Abeta42 (cholera toxin B subunit and Abeta42 fusion protein) in vivo. A single administration of the AAV-CB-Abeta42 vaccine induced a prolonged, strong production of Abeta-specific serum IgG in transgenic mice that overexpressed the London mutant of amyloid precursor protein (APP/V717I), and resulted in improved ability of memory and cognition, decreased Abeta deposition in the brain, and a resultant decrease in plaque-associated astrocytosis. Our results extended the immunological approaches for the treatment and prevention of AD to an oral, intranasal, or intramuscular route that might be better tolerated in human patients than repetitive parental immunizations in the presence of adjuvant. AAV has attracted tremendous interest as a promising vector for gene delivery. Our results raised the possibility that AAV-CB-Abeta42 vector immunization may provide the basis of a novel and promising Alzheimer's disease vaccination program.