Evaluation and optimization of immunomagnetic methods for the purification and selection of CMV-specific cytotoxic T lymphocytes from G-CSF primed peripheral blood mononuclear cells for adoptive immunotherapy

Adoptive transfer of cytomegalovirus (CMV)-specific T cells has shown promising results in preventing pathological effects caused by opportunistic CMV infection in patients following allogeneic hematopoietic stem cell transplantation. The majority of studies have used steady-state leukapheresis for CMV-reactive product manufacture, a collection obtained prior to or weeks after granulocyte-colony stimulating factor (G-CSF) mobilization, but the procurement of this additional sample is often not available in the unrelated donor setting. If the cellular product for adoptive immunotherapy could be generated from the same G-CSF mobilized collection, the problems associated with the additional harvest could be overcome. However, the tolerogenic effects induced by G-CSF on T cells could affect the anti-viral in vivo functionality of CMV-specific T cells obtained from G-CSF mobilized donor samples if they were infused into the patient. In this thesis it has been investigated the use of G-CSF mobilized samples for the manufacture of CMV-specific T cells using two direct selection methods. First, CMV-specific T cells were isolated based on activation-dependent CD137 expression, which demonstrated a high specificity for CMV, the secretion of cytotoxic effector molecules, proliferation in response to the antigen and lysis of CMV-loaded target cells, showing that they shared the same characteristics as CMV-specific T cells obtained from non-mobilized cells. On the other hand, CMV-specific CD8+ T cells were isolated using MHC-multimers and showed that products obtained from G-CSF mobilized samples were able to respond to the antigen by expressing activation markers and secreting pro-inflammatory cytokines and lytic molecules, but the in vitro proliferative capacity was impaired

Evaluation and optimization of immunomagnetic methods for the purification and selection of CMV-specific cytotoxic T lymphocytes from G-CSF primed peripheral blood mononuclear cells for adoptive immunotherapy

Adoptive transfer of cytomegalovirus (CMV)-specific T cells has shown promising results in preventing pathological effects caused by opportunistic CMV infection in patients following allogeneic hematopoietic stem cell transplantation. The majority of studies have used steady-state leukapheresis for CMV-reactive product manufacture, a collection obtained prior to or weeks after granulocyte-colony stimulating factor (G-CSF) mobilization, but the procurement of this additional sample is often not available in the unrelated donor setting. If the cellular product for adoptive immunotherapy could be generated from the same G-CSF mobilized collection, the problems associated with the additional harvest could be overcome. However, the tolerogenic effects induced by G-CSF on T cells could affect the anti-viral in vivo functionality of CMV-specific T cells obtained from G-CSF mobilized donor samples if they were infused into the patient. In this thesis it has been investigated the use of G-CSF mobilized samples for the manufacture of CMV-specific T cells using two direct selection methods. First, CMV-specific T cells were isolated based on activation-dependent CD137 expression, which demonstrated a high specificity for CMV, the secretion of cytotoxic effector molecules, proliferation in response to the antigen and lysis of CMV-loaded target cells, showing that they shared the same characteristics as CMV-specific T cells obtained from non-mobilized cells. On the other hand, CMV-specific CD8+ T cells were isolated using MHC-multimers and showed that products obtained from G-CSF mobilized samples were able to respond to the antigen by expressing activation markers and secreting pro-inflammatory cytokines and lytic molecules, but the in vitro proliferative capacity was impaired

The Induction of Cytokine Release in Monocytes by Electronegative Low-Density Lipoprotein (LDL) Is Related to Its Higher Ceramide Content than Native LDL

Abstract: Electronegative low-density lipoprotein (LDL(−)) is a minor modified LDL subfraction that is present in blood. LDL(−) promotes inflammation and is associated with the development of atherosclerosis. We previously reported that the increase of cytokine release promoted by this lipoprotein subfraction in monocytes is counteracted by high-density lipoprotein (HDL). HDL also inhibits a phospholipase C-like activity (PLC-like) intrinsic to LDL(−). The aim of this work was to assess whether the inhibition of the PLC-like activity by HDL could decrease the content of ceramide (CER) and diacylglycerol (DAG) generated in LDL(−). This knowledge would allow us to establish a relationship between these compounds and the inflammatory activity of LDL(−). LDL(−) incubated at 37 °C for 20 h increased its PLC-like activity and, subsequently, the amount of CER and DAG. We found that incubating LDL(−) with HDL decreased both products in LDL(−). Native LDL was modified by lipolysis with PLC or by incubation with CER-enriched or DAG-enriched liposomes. The increase of CER in native LDL significantly increased cytokine release, whereas the enrichment in DAG did not show these inflammatory properties. These data point to CER, a resultant product of the PLC-like activity, as a major determinant of th

Effect of CD25 immunomagnetic depletion on CD25+ CMVpp65 CMV-T and CD25+ FoxP3+ Tregs.

<p>CD25 expression was assessed in paired unmanipulated and CD25-depleted G-CSF-mobilized PBMCs following CMVpp65 peptide stimulation (A). FoxP3 expression was analysed both pre and post CD25-enrichment amongst CD4+ T cells (B–D) and CD25 purity and yield determined following CD25 enrichment (D). **<i>p</i><0.01 and *<i>p</i><0.05 in a paired <i>t</i> test. Neg, negative.</p

Qualitative and quantitative assessment of cytokine secretion by expanded cells after antigenic re-challenge.

<p>Expanded CD25+ cells isolated from both G-CSF-mobilized and non-mobilized donors were stimulated with CMVpp65 peptide loaded autologous PBMCs in the presence of Brefeldin A and purified anti CD28-antibody. Histograms illustrate CMVpp65 rechallenge experiments in representative CD25+ expansions from a G-CSF-mobilized and non-mobilized donor, analysed for IL-2, TNF-α, IFN-γ, Granzyme B and IL-10. Open peaks represent isotype matched controls (A). The combined assessment in G-CSF-mobilized (<i>n</i> = 5) and non-mobilized (<i>n</i> = 5) donors are summarized (B). **<i>p</i><0.01, in an unpaired <i>t</i> test. Gra, granzyme; Mob, mobilized.</p

Identification and Isolation of CD25+ CMVpp65-specific T cells in G-CSF-mobilized and non-mobilized PBMCs.

<p>G-CSF-mobilized (<i>n</i> = 6) and non-mobilized PBMCs (<i>n</i> = 5) were stimulated for 24 hours or left untouched (negative control) and samples taken at 1, 4, 6, 16 and 24 hours for analysis of CD25 expression amongst CD3+ T cells (A). Purity and yield of CD25 expressing cells was determined from within the CD3+ population following selection (B). Evaluation of FoxP3 expression in CD4+ CD25+ T cells in G-CSF-mobilized and non-mobilized PBMCs was assessed in pre-stimulated, CMVpp65 stimulated and post CD25+ selection after gating on CD3+ CD4+ T cells (C). Cumulative data of FoxP3 expression was assessed by analysing CD25+FoxP3+ and CD25+FoxP3− populations per 10,000 CD4+ T cells in both G-CSF-mobilized and non-mobilized PBMCs (D). **<i>p</i><0.01 and *<i>p</i><0.05 in an unpaired <i>t</i> test comparing the absolute number of CD25+FoxP3+ cells. Stim, stimulated; Mob, mobilized; N-Mob, non-mobilized; Neg, negative.</p

Assessment of FoxP3 expression and suppressive capacity of CD25+ expanded cells in G-CSF-mobilized PBMCs.

<p>FoxP3 expression was assessed in G-CSF-mobilized CD25+ expanded cells after 14–24 days in culture. FACS plots illustrate results in 3 expansion experiments (A). FoxP3 expression was compared between CD25+ expanded cells (<i>n</i> = 5) and CD25+ fractions following magnetic enrichment (<i>n</i> = 5) (B). Suppression of CFSE-labelled PBMCs proliferation by CD25+ expanded cells (<i>n</i> = 3) was determined at three ratios in the presence of anti-CD3 antibody cultured for 5 days (C). PBMCs alone (CFSE) and PBMCs incubated with anti-CD3 antibody were cultured as experimental controls. Assessment of suppressive capacity was determined by comparing CD25+ expanded cells with CD25+ fractions (D). ***<i>p</i><0.0001, in an unpaired <i>t</i> test.</p

Expansion of CD25+ CMVpp65-specific T cells in short term culture.

<p>0.25×10⁶ CD25+ T cells were expanded in culture for up to 24 days in the presence of IL-7, IL-15 and 12.5×10⁶ irradiated autologous feeder cells (ratio of 1∶50) in G-CSF-mobilized and non-mobilized donors. CD25+ expanded cells were analysed for the proportion of CD8+ (open bars) and CD4+ (grey bars) T cells amongst CD3+ T cells in both G-CSF-mobilized and non-mobilized donors (A). Expanded cells were assessed for co-expression of CD25 and CD69 following CMVpp65 re-challenge in both G-CSF-mobilized (<i>n = 5</i>) and non-mobilized (<i>n = 5</i>) expansions (B). Expanded cells were also challenged with control feeders (without CMVpp65 peptides). G-Mob, G-CSF-Mobilized; Non-mob, Non-Mobilized.</p