Fenlu Zhu

Functionally distinct helper T-cell epitopes of HCV and their role in modulation of NS3-specific, CD8+/tetramer positive CTL

Hepatitis C virus specific (HCV-specific) CD8+ cytotoxic T cells play a critical role in viral clearance. Low HCV-specific cytotoxic T lymphocyte (CTL) responses in chronic HCV infection may favor the persistence of virus, whereas stimulation and expansion of HCV-specific CTL activity may assist elimination of HCV infection. Helper T cells control the intensity of CD8+ T-cell responses and helper T-cell responses are known to be compromised in chronic carriers of HCV. In this study, we wanted to ascertain if strengthening the Th response could increase the intensity of CTL activity against HCV target antigens. We selected a synthetic CTL peptide NS3(1073-1081)), two Th1 epitopes, peptide NS3(358-375) and NS5B(155-172), and one Th2 epitope, peptide NS3(505-521). By using the four peptides alone or in combinations, we stimulated peripheral blood cells isolated from a chronic hepatitis C patient in vitro and then analyzed CD8 T cells specific for the NS3(1073-1081) CTL epitope in A2 tetramer staining and cytotoxicity assays. The results demonstrated that CTL responses could be augmented by helper T-cell epitopes NS3(358-375) and NS5B(155-172). Th2 epitope NS3(505-521) inhibited augmentation of CTL activity by Th1 epitopes. This inhibitory effect could be overcome by combining the two Th1 epitopes NS3(358-375) and NS5B(155-172) together with NS3(505-521). Under such conditions, CTL frequency was restored, but cytotoxic activity remained low suggesting that the help provided under these cultures was sufficient to drive proliferation of CTL, but not sufficient to drive differentiation into mature killer cells. These results may provide some insights into compromised CTL activity in HCV viral persistence.

Stimulation by means of dendritic cells followed by Epstein-Barr virus-transformed B cells as antigen-presenting cells is more efficient than dendritic cells alone in inducing Aspergillus f16-specific cytotoxic T cell responses.

Adoptive immunotherapy with in vitro expanded antigen‐specific cytotoxic T lymphocytes (CTLs) may be an effective approach to prevent, or even treat, Aspergillus (Asp) infections. Such lines can be generated using monocyte‐derived dendritic cells (DC) as antigen‐presenting cells (APC) but requires a relatively high volume of starting blood. Here we describe a method that generates Asp‐specific CTL responses more efficiently using a protocol of antigen presented on DC followed by Epstein–Barr virus (EBV)‐transformed B lymphoblastoid cell lines (BLCL) as APC. Peripheral blood mononuclear cells were stimulated weekly (2–5×) with a complete pool of pentadecapeptides (PPC) spanning the coding region of Asp f16 pulsed onto autologous mature DC. Cultures were split and stimulated subsequently with either PPC‐DC or autologous PPC‐pulsed BLCL (PPC‐BLCL). Lines from the DC/BLCL arm demonstrated Asp f16‐specific cytotoxicity earlier and to a higher degree than lines generated with PPC‐DC alone. The DC/BLCL‐primed lines showed a higher frequency of Asp f16‐specific interferon (IFN)‐γ producing cells but an identical effector cell phenotype and peptide specificity compared to PPC‐DC‐only‐primed lines. Tumour necrosis factor (TNF)‐α, but not IL‐10, appeared to play a role in the effectiveness of BLCL as APC. These results demonstrate that BLCL serve as highly effective APC for the stimulation of Asp f16‐specific T cell responses and that a culture approach using initial priming with PPC‐DC followed by PPC‐BLCL may be a more effective method to generate Asp f16‐specific T cell lines and requires less starting blood than priming with PPC‐DC alone.

Hydroxyethyl starch as a substitute for dextran 40 for thawing peripheral blood progenitor cell products

BACKGROUND AIMS Removing DMSO post-thaw results in: reduced infusion reactions, improved recovery and stability of viable CD34+ cells. Validated methods use 5%-8.3% Dextran 40 with 2.5%-4.2% HSA for this purpose. Recent shortages of clinical grade Dextran require identification of suitable alternatives. METHODS PBPC were used to compare a standard 2X wash medium of 5 parts 10% Dextran 40 in saline (DEX) with 1 part 25% HSA (8.3% DEX/ 4.2% HSA) with Hydroxyethyl Starch (HES)-based solutions. Cells in replicate bags were diluted with an equal volume of wash solution, equilibrated 5 minutes, the bag filled with wash medium, pelleted and the supernatant expressed. Bags were restored to the frozen volume in wash medium and tested by single platform flow cytometry and CFU. Total viability, viable TNC, MNC, and CD34+ cell recovery, and CD34+ cell viability were compared immediately post-thaw and after 90 minutes. RESULTS 5.2% HES/4.2% HSA did not differ from our standard in CD34 recovery or viability. Due to concerns that high concentrations of HES could affect renal function we tested 0.6% HES/2.5% HSA resulting in significantly poorer CD34 recovery and viability. Results improved using 2.4% HES/4.2% HSA and when 0.6% HES/4.2%HSA was used no significant differences were seen. CFU assays confirmed no differences between the standard dextran arm and HES at 2.4% or 0.6% so long as HSA was at 4.2%. CONCLUSIONS We conclude that HES from 0.6% to 5.2% with 4.2% HSA is a suitable substitute for Dextran 40 as a reconstitution/washing medium for PBPC products.

Generation of cytotoxic T-cell lines using overlapping pentadecapeptides derived from conserved regions of the adenovirus hexon protein.

Many of the 51 serotypes of adenovirus have been associated with clinically relevant infection. Adenovirus can disseminate rapidly in patients with a compromised immune system, such as that which occurs secondary to haematopoietic progenitor-cell transplantation. The higher rate of infection in recipients of T cell-depleted grafts and in those undergoing T cell-targeted treatment during graft versus host disease demonstrates the importance of a T-cell response in preventing disseminated infection. Studies have shown that the memory response to adenovirus is directed primarily to the hexon protein and is dominated by CD4+ T cells, probably due to the ability of the virus to block its presentation on HLA class I antigens. We have developed an approach to expand adenovirus-specific T cells using a pool of overlapping pentadecapeptides derived from selected conserved regions of hexon. We characterized responses to identify the peptides that are recognized, the responding T-cell subsets and their HLA restriction. Of eight lines that were characterized extensively, seven included both CD4+ and CD8+ T cells and each recognized between two and eight unique peptide sequences. By focusing the response on the conserved sequences of hexon, the cell lines are likely to recognize most of the serotypes responsible for clinically relevant disease. The 15 aa peptides used to prime the responses are more likely than whole virus or longer peptides to expand the less frequent CD8+ memory subset. Lines prepared by using our method may be more effective in adoptive immunotherapy protocols designed to prevent or treat disseminated adenovirus infections in high-risk patients.

A colitogenic memory CD4+ T cell population mediates gastrointestinal graft-versus-host disease

Damage to the gastrointestinal tract is a major cause of morbidity and mortality in graft-versus-host disease (GVHD) and is attributable to T cell-mediated inflammation. In this work, we identified a unique CD4+ T cell population that constitutively expresses the β2 integrin CD11c and displays a biased central memory phenotype and memory T cell transcriptional profile, innate-like properties, and increased expression of the gut-homing molecules α4β7 and CCR9. Using several complementary murine GVHD models, we determined that adoptive transfer and early accumulation of β2 integrin-expressing CD4+ T cells in the gastrointestinal tract initiated Th1-mediated proinflammatory cytokine production, augmented pathological damage in the colon, and increased mortality. The pathogenic effect of this CD4+ T cell population critically depended on coexpression of the IL-23 receptor, which was required for maximal inflammatory effects. Non-Foxp3-expressing CD4+ T cells produced IL-10, which regulated colonic inflammation and attenuated lethality in the absence of functional CD4+Foxp3+ T cells. Thus, the coordinate expression of CD11c and the IL-23 receptor defines an IL-10-regulated, colitogenic memory CD4+ T cell subset that is poised to initiate inflammation when there is loss of tolerance and breakdown of mucosal barriers.

Selection of trkB-binding peptides from a phage-displayed random peptide library

Brain-derived neurotrophic factor (BDNF) shows potential in the treatment of neurodegenerative diseases, but the therapeutic application of BDNF has been greatly limited because it is too large in molecular size to permeate blood-brain barrier. To develop low-molecular-weight BDNF-like peptides, we selected a phage-displayed random peptide library using trkB expressed on NIH 3T3 cells as target in the study. With the strategy of peptide library incubation with NIH 3T3 cells and competitive elution with 1 μg/mL of BDNF in the last round of selection, the specific phages able to bind to the natural conformation of trkB and antagonize BDNF binding to trkB were enriched effectively. Five trkB-binding peptides were obtained, in which a core sequence of CRA/TXΦXXΦXXC (X represents the random amino acids, Φ represents T, L or I) was identified. The BDNF-like activity of these five peptides displayed on phages was not observed, though all of them antagonized the activity of BDNF in a dose-dependent manner. Similar results were obtained with the synthetic peptide of C1 clone, indicating that the 5 phage-derived peptides were trkB antagonists. These low-molecular-weight antagonists of trkB may be of potential application in the treatment of neuroblastoma and chronic pain. Meanwhile, the obtained core sequence also could be used as the base to construct the secondary phage-displayed peptide library for further development of small peptides mimicking BDNF activity.

Tocilizumab, tacrolimus and methotrexate for the prevention of acute graft-versus-host disease: low incidence of lower gastrointestinal tract disease

We conducted a phase 2 study in which patients undergoing allogeneic hematopoietic stem cell transplantation received tocilizumab in addition to standard immune suppression with tacrolimus and methotrexate for graft-versus-host disease prophylaxis. Thirty-five patients were enrolled between January 2015 and June 2016. The median age of the cohort was 66 (range: 22-76). All patients received busulfan-based conditioning, and were transplanted with human leukocyte antigen-matched related or matched unrelated bone marrow or peripheral stem cell grafts. The cumulative incidences of grades II-IV and III-IV acute graft-versus-host disease were 14% (95% CI 5-30) and 3% (95% CI 0-11) at day 100, and 17% (95% CI 7-31) and 6% (95% CI 1-16) at day 180, respectively. Notably, there were no cases of graft-versus-host disease of the lower gastrointestinal tract within the first 100 days. A comparison to 130 matched controls who only received tacrolimus and methotrexate demonstrated a lower cumulative incidence of grades II-IV acute graft-versus-host disease (17% versus 45%, P=0.003) and a significant increase in grades II-IV acute graft-versus-host disease-free survival at six months (69% versus 42%, P=0.001) with tocilizumab, tacrolimus and methotrexate, which was the primary endpoint of the study. Immune reconstitution was preserved in patients treated with tocilizumab, tacrolimus and methotrexate, as T-cell and B-cell subsets recovered to near normal levels by 6-12 months post-transplantation. We conclude that tocilizumab has promising activity in preventing acute graft-versus-host disease, particularly in the lower gastrointestinal tract, and warrants examination in a randomized setting.

Closed-system manufacturing of CD19 and dual-targeted CD20/19 chimeric antigen receptor T cells using the CliniMACS Prodigy device at an academic medical center

BACKGROUND AIMS Multiple steps are required to produce chimeric antigen receptor (CAR)-T cells, involving subset enrichment or depletion, activation, gene transduction and expansion. Open processing steps that increase risk of contamination and production failure are required. This complex process requires skilled personnel and costly clean-room facilities and infrastructure. Simplified, reproducible CAR-T-cell manufacturing with reduced labor intensity within a closed-system is highly desirable for increased availability for patients. METHODS The CliniMACS Prodigy with TCT process software and the TS520 tubing set that allows closed-system processing for cell enrichment, transduction, washing and expansion was used. We used MACS-CD4 and CD8-MicroBeads for enrichment, TransAct CD3/CD28 reagent for activation, lentiviral CD8 TM-41BB-CD3 ζ-cfrag vectors expressing scFv for CD19 or CD20/CD19 antigens for transduction, TexMACS medium-3%-HS-IL2 for culture and phosphate-buffered saline/ethylenediaminetetraacetic acid buffer for washing. Processing time was 13 days. RESULTS Enrichment (N = 7) resulted in CD4/CD8 purity of 98 ± 4.0%, 55 ± 6% recovery and CD3+ T-cell purity of 89 ± 10%. Vectors at multiplicity of infection 5-10 resulted in transduction averaging 37%. An average 30-fold expansion of 108 CD4/CD8-enriched cells resulted in sufficient transduced T cells for clinical use. CAR-T cells were 82-100% CD3+ with a mix of CD4+ and CD8+ cells that primarily expressed an effector-memory or central-memory phenotype. Functional testing demonstrated recognition of B-cells and for the CAR-20/19 T cells, CD19 and CD20 single transfectants were recognized in cytotoxic T lymphocyte and interferon-γ production assays. DISCUSSION The CliniMACS Prodigy device, tubing set TS520 and TCT software allow CAR-T cells to be manufactured in a closed system at the treatment site without need for clean-room facilities and related infrastructure.