Individual variation in the frequency of HLA class II‐specific cytotoxic T lymphocyte precursors

Man, Stephen; Lechler, Robert I.; Batchelor, J. R.; Sharrock, C.

doi:10.1002/eji.1830200420

Cited by 22 publications

(14 citation statements)

References 43 publications

(23 reference statements)

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“…This suggests that both T-cell subsets can be redirected to become cytolytic against CLL cells in the presence of blinatumomab. Although cytotoxic CD4 + T cells are relatively rare in healthy subjects, 30 an expanded population of CD4 + perforin + T-cells with an EM phenotype has been described in CLL patients. 31 Our study used 3-or 7-day cultures of PBMC treated with blinatumomab.…”

Section: Discussionmentioning

confidence: 99%

Blinatumomab induces autologous T-cell killing of chronic lymphocytic leukemia cells

et al. 2013

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© F e r r a t a S t o r t i F o u n d a t i o n 2 0 1 3of CLL activation and pro-survival signals. Given these data, this approach represents a novel and promising therapeutic strategy for the treatment of CLL and, potentially, other lymphoid malignancies. Methods Blood samples from patients with chronic lymphocytic leukemiaTwenty-eight CLL patients (aged 53-83 years) were studied with full ethical approval. All blood samples from patients were obtained with informed consent (see Online Supplementary Methods). The majority of the assays were performed on fresh samples from treatment-naïve patients, except those for which the results are shown in Figure 1: in these assays, samples from patients who had received standard chemotherapy treatment were also tested. Antibodies and flow cytometryA full list of the antibodies used can be found in the Online Supplementary Methods. Blinatumomab (MT103, AMG103) was provided by Amgen Inc. (Munich, Germany). T cells were identified using CD4 and CD8 markers, while CLL cells were CD5 + CD4-CD8 -(>99% CD19 + ). Cell culturesPeripheral blood mononuclear cells (PBMC) were incubated in RPMI 1640 supplemented with 5% AB serum (AB media) for 3-7 days. Blinatumomab was added to PBMC cultures at a concentration of 10 or 100 ng/mL. Human T-cell activator CD3/CD28 dynabeads (Invitrogen) were added at 1x10 5 beads/1x10 6 PBMC as a positive control for T-cell activation. For absolute counts of T cells and CLL cells, an anti-CD3 antibody (Invitrogen) at a dose of 27.7 or 277 ng/mL was used as a positive control (same molar concentration as 10 or 100 ng/mL blinatumomab). Intracellular Ki67 stainingPBMC were labeled with antibodies against CD4, CD8, CCR7 and CD45RA, fixed and permeabilized (Fix and Perm with 1% NP40, Caltag-Medsystems, Buckingham, UK) before staining with a Ki67-FITC antibody for flow cytometric analysis. Cytokine secretion assayCytokines in tissue culture supernatants were measured using a Human Th1/Th2 11plex RTU Flowcytomix kit (eBioscience) for simultaneous detection of 11 cytokines. Intracellular cytokine staining and the determination of CD107 and granzyme B expressionPBMC were cultured (3 days) with 10 ng/mL blinatumomab, before treatment with Golgi plug and Golgi stop (BD biosciences), and incubation with an anti-CD107 antibody (5 h at 37°C). Intracellular expression of granzyme B or interferon (IFN)-g and tumor necrosis factor (TNF)-α in CD4 or CD8 T cells was measured by flow cytometry. Absolute counts of T cells and chronic lymphocytic leukemia cellsPBMC samples that had or had not been treated with blinatumomab (7 days) were surface-stained with antibodies against annexin V, CD5, CD8 and CD4. Absolute counts were determined by flow cytometry using Cytocount beads (Dako, Stockport, UK). Flow cytometry-based cytotoxicity assayMouse fibroblast cells transfected with CD40L [TL(CD40L)] and non-transfected cells (NTL) were cultured as previously described. 21 For co-cultures, irradiated (80 Gy) NTL or TL(CD40L) cells were seeded into a 48-well plate, and allow...

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Section: Discussionmentioning

confidence: 99%

Blinatumomab induces autologous T-cell killing of chronic lymphocytic leukemia cells

et al. 2013

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“…Over the past 2 decades, the ability of CD4 ϩ T cells to display cytotoxic potential has been reported in isolated, but repeated, reports in both mouse and human (2)(3)(4)(5)(6). However, the observation of such cytotoxic activity in CD4 ϩ T cells has usually been restricted to cell lines and CD4 ϩ T cell clones generated by long term in vitro culture and has therefore been considered by some authors to be an artifact (7).…”

Section: T He Cd4mentioning

confidence: 99%

Characterization of CD4+ CTLs Ex Vivo

Appay

Zaunders

Papagno

et al. 2002

The Journal of Immunology

488

460

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The cytotoxic potential of CD8+ T cells and NK cells plays a crucial role in the immune response to pathogens. Although in vitro studies have reported that CD4+ T cells are also able to mediate perforin-mediated killing, the in vivo existence and relevance of cytotoxic CD4+ T cells have been the subject of debate. Here we show that a population of CD4+ perforin+ T cells is present in the circulation at low numbers in healthy donors and is markedly expanded in donors with chronic viral infections, in particular HIV infection, at all stages of the disease, including early primary infection. Ex vivo analysis shows that these cells have cytotoxic potential mediated through the release of perforin. In comparison with more classical CD4+ T cells, this subset displays a distinct surface phenotype and functional profile most consistent with end-stage differentiated T cells and include Ag experienced CD4+ T cells. The existence of CD4+ cytotoxic T cells in vivo at relatively high levels in chronic viral infection suggests a role in the immune response.

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“…Autologous CD8 ϩ -enriched responder T lymphocytes were prepared from the nonadherent cell fraction obtained on day 0 by immunomagnetic beads (Dynal Biotech, Wirral, U.K.) and depletion of CD4 ϩ and CD16 ϩ cells (37), and then cryopreserved. On day 6, the cells were thawed and added at 2 ϫ 10 6 cells/well to the adenovirus-infected DCs (for a responder-stimulator ratio of 20:1) in 1 ml/well RPMI/AB supplemented with human rIL-7 (Genzyme, Cambridge, MA) at a final concentration of 5 ng/ml.…”

Section: In Vitro Ctl Induction Using Dcsmentioning

confidence: 99%

Antigen Processing Defects in Cervical Carcinomas Limit the Presentation of a CTL Epitope from Human Papillomavirus 16 E6

Evans

Borysiewicz

Evans

et al. 2001

The Journal of Immunology

Self Cite

100

114

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Human papillomavirus (HPV) infection, particularly type 16, is causally associated with the development of cervical cancer. The E6 and E7 proteins of HPV are constitutively expressed in cervical carcinoma cells making them attractive targets for CTL-based immunotherapy. However, few studies have addressed whether cervical carcinomas can process and present HPV E6/E7-derived Ags for recognition by CTL. We generated HLA-A*0201-restricted CTL clones against HPV16 E629–38 that recognized HPV16 E6 Ags transfected into B lymphoblastoid cells. These CTL were unable to recognize HLA-A*0201+ HPV16 E6+ cervical carcinoma cell lines even when the level of endogenous HPV16 E6 in these cells was increased by transfection. This defect in presentation of HPV16 E629–38 correlated with low level expression of HLA class I, proteasome subunits low molecular mass protein 2 and 7, and the transporter proteins TAP1 and TAP2 in the cervical carcinoma cell lines. The expression of all of these proteins could be up-regulated by IFN-γ, but this was insufficient for CTL recognition unless the level of HPV16 E6 Ag was also increased by transfection. CTL recognition of the HPV16 E629–38 epitope in 721.174 B cells was dependent on TAP expression but independent of immunoproteasome expression. Collectively, these findings suggest that presentation of the HPV16 E629–38 epitope in cervical carcinoma cell lines is limited both by the level of TAP expression and by the low level or availability of the source HPV E6 oncoprotein. These observations place constraints on the use of this, and potentially other, HPV-derived CTL epitopes for the immunotherapy of cervical cancer.

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Individual variation in the frequency of HLA class II‐specific cytotoxic T lymphocyte precursors

Cited by 22 publications

References 43 publications

Blinatumomab induces autologous T-cell killing of chronic lymphocytic leukemia cells

Blinatumomab induces autologous T-cell killing of chronic lymphocytic leukemia cells

Characterization of CD4+ CTLs Ex Vivo

Antigen Processing Defects in Cervical Carcinomas Limit the Presentation of a CTL Epitope from Human Papillomavirus 16 E6

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