Inactivation of Human Leukocytes in Platelet Products After Pathogen Reduction Technology Treatment in the Presence of Platelet Additive Solution.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2116-2116
Author(s):  
Loren D. Fast ◽  
Susanne Marschner ◽  
Gilbert DiLeone ◽  
Raymond Goodrich
Keyword(s):  
T Cell Activation ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Uv Light ◽  
Human Peripheral Blood ◽  
Control Sample ◽  
Blood Products ◽  
Peripheral Blood Mononuclear ◽  
Pathogen Reduction ◽  
Additive Solution

Abstract Abstract 2116 Poster Board II-93 During the transfusion of blood or blood products, a recipient can receive a large number of allogeneic leukocytes. This can lead to leukocyte-mediated adverse reactions in the recipient and include donor anti-recipient responses such as the life-threatening transfusion-associated graft versus host disease (TA-GVHD) and cytokine production; or recipient anti-donor responses that are induced by direct presentation of foreign antigen by donor leukocytes or indirectly after processing of the donor cells by recipient antigen-presenting cells. To avoid or minimize leukocyte mediated reactions, the leukocytes present in blood products are inactivated or depleted prior to administration. Nucleic acid targeted pathogen reduction processes (PRT) are well suited for leukocyte inactivation. The Mirasol® PRT System uses riboflavin (Vitamin B2) and ultraviolet (UV) light to reduce the active pathogen load and inactivate residual leukocytes in blood products used for transfusion. To make the PRT System more widely applicable, the effect of treating leukocytes in the presence of platelet additive solution (PAS) was tested. Human peripheral blood mononuclear cells (PBMNC) were purified by Ficoll-Hypaque discontinuous centrifugation and placed in 350 ml of storage solution consisting of 65% PAS (SSP+) and 35% plasma. An untreated control sample was removed before addition of 35 ml of riboflavin (500 μM) and exposure to UV light (9.1 J/ml). PBMNC were recovered after treatment and tested for their ability to proliferate in response to polyclonal stimulators such as phytohemagglutinin, and anti-CD3/CD28 or to allogeneic stimulator cells in a mixed lymphocyte culture (MLC). Treatment was found to inhibit proliferation as well as T cell activation as measured by the upregulation of CD69 expression when incubated with phorbol 12-myristate 13-acetate. Treated PBMNC were unable to produce inflammatory or TH1/TH2 cytokines when stimulated with lipopolysaccharide for 24 hours or anti-CD3/CD28 for 72 hours. Levels of cytokines that are released in the absence of activation, such as IL-6, IL-8 and IL1β, were reduced below levels of detection of the assay after PRT-treatment. Quantitation of the degree of inactivation using limiting dilution assays showed that 5.2 log inactivation could be achieved at the specified energy doses. These treatment conditions resulted in acceptable platelet cell quality over 8 days in storage. In summary, PRT treatment was able to functionally inactivate leukocytes in the presence of PAS to the levels seen with gamma-irradiation without adversely affecting the quality of the platelets. Disclosures: Fast: CaridianBCT Biotechnologies: Research Funding. Marschner:CaridianBCT Biotechnologies: Employment. Goodrich:CaridianBCT Biotechnologies: Employment.

PBMC-07- Stimulation of human peripheral blood mononuclear cells with immobilized anti-CD3 and soluble anti-CD28 antibodies. v1

2021 ◽  
Author(s):  
Alessia Furgiuele ◽  
Massimilano Legnaro ◽  
Alessandra Luini ◽  
Marco Ferrari ◽  
Emanuela Rasini ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Functional Responses ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

This protocol was designed to activate the lymphocytes T of a population of peripheral blood mononuclear cells (PBMCs), simulating their physiological response to antigen/MHC complex acting on T Cell Receptors-TCR , in order to test their functional responses including cell proliferation and cytokine production. The co-stimulation protocol include: i)anti-CD3 antibody a polyclonal activator specific for invariant framework epitopes on TCR complex (in particular, we use UCHT1 clone an anti-human CD3 antibody that recognizes the ε-chain of CD3 which is used for immobilized option of activation) (http://static.bdbiosciences.com/documents/BD_Tcell_Human_CD3_Activation_Protocol.pdf) ii) anti-CD28 antibody used to cooperate with TCR signals promoting activation of T cells The procedure has been reproduced following the indications contained in the protocol of "EBiooscience" (https://tools.thermofisher.com/content/sfs/manuals/t-cell-activation-in-vitro.pdf). Pilot experiments on PBMC were carried out to determine the best concentrations of anti-CD3 and anti-CD28 to induce optimal proliferation of PBMC and production of cytokines TNF-α and IFN-γ. We found a dose dependent correlation between immobilized anti-CD3 and cells functional responses. The selected amount was 2 µg/mL for both anti-CD3 and anti-CD28 that was the concentration below the maximum response which allows also to test possible modulations by therapeutic agents. References http://static.bdbiosciences.com/documents/BD_Tcell_Human_CD3_Activation_Protocol.pdf https://tools.thermofisher.com/content/sfs/manuals/t-cell-activation-in-vitro.pdf https://www.bdbiosciences.com/ds/pm/tds/555330.pdf https://www.bdbiosciences.com/ds/pm/tds/555726.pdf BEFORE STARTING with this procedure Moreover, work under laminar flow hood when you are processing samples from the beginning to the end of the culture. Make sure you are using,sterile culture mediumand sterile plastic disposable as well.

Inactivation of Human White Blood Cells in Red Blood Cell Products Using the MIRASOL® System for Whole Blood.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2897-2897
Author(s):  
Loren D. Fast ◽  
Susanne Marschner ◽  
Gilbert DiLeone ◽  
Suzann Doane ◽  
Christy Fitzpatrick ◽  
...  
Keyword(s):  
Immune Responses ◽  
T Cell Activation ◽  
Whole Blood ◽  
Blood Cells ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Protein Quality ◽  
Human Peripheral Blood ◽  
White Blood Cells ◽  
Blood Products

Abstract Transfusion of blood products containing white blood cells (WBC) can result in the induction of immune responses that can negatively impact the recipient. An approach that would mitigate these consequences would be beneficial. Previous studies had shown that exposure of platelet concentrates to light in the presence of riboflavin was able to inhibit immune responses mediated by WBC. To make this protocol more widely applicable the effect of treating whole blood units with riboflavin and varying amounts of light was tested. Human peripheral blood mononuclear cells were purified by Ficoll-Hypaque discontinuous centrifugation from aliquots of nonleukoreduced whole blood units that were untreated or exposed to Mirasol treatment using varying light dosages. Viability and phenotype of treated cells was unchanged compared to untreated controls. The results showed that exposure of whole blood units to 33J/mL red blood cells (RBC) UV light in the presence of riboflavin completely inhibited proliferation of WBC in response to polyclonal stimulators such as phytohemagglutinin, and anti-CD3/CD28 or to allogeneic stimulator cells in a mixed lymphocyte culture (MLC). Additional assays showed that treated WBC were unable to induce proliferation of normal responder cells in an MLC. Treated cells did not produce inflammatory or TH1/TH2 cytokines when stimulated with lipopolysaccharide for 24 hours or anti-CD3/CD28 for 72 hours. In addition, treatment was found to inhibit T cell activation as evidenced by the lack of CD69 expression in treated compared to untreated control cells when incubated with phorbol 12-myristate 13-acetate. These treatment conditions did not induce crossmatch incompatibility. Methemoglobin levels and hemolysis in RBC units stayed below 1% during storage for 42 days in AS-3. Platelet and plasma units separated from whole blood after treatment showed acceptable cell and protein quality over 5 days in storage or as fresh frozen plasma, respectively. In summary, Mirasol treatment was able to functionally inactivate WBC in whole blood products without adversely affecting the quality of the RBC, platelets and plasma. This technique offers potential means to achieve inactivation of WBC in whole blood units that can subsequently be separated into RBC, platelet and plasma components.

scholarly journals Endogenous retrovirus-encoded Syncytin-2 contributes to exosome-mediated immunosuppression of T cells†

2019 ◽  
Author(s):  
Adjimon G Lokossou ◽  
Caroline Toudic ◽  
Phuong Trang Nguyen ◽  
Xavier Elisseeff ◽  
Amandine Vargas ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell Activation ◽  
Map Kinases ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Endogenous Retrovirus ◽  
Jurkat Cells ◽  
Peripheral Blood Mononuclear ◽  
Interfering Rna

Abstract Modulation of the activation status of immune cell populations during pregnancy depends on placental villous cytotrophoblast (VCT) cells and the syncytiotrophoblast (STB). Failure in the establishment of this immunoregulatory function leads to pregnancy complications. Our laboratory has been studying Syncytin-2 (Syn-2), an endogenous retroviral protein expressed in placenta and on the surface of placental exosomes. This protein plays an important role not only in STB formation through its fusogenic properties, but also through its immunosuppressive domain (ISD). Considering that Syn-2 expression is importantly reduced in preeclamptic placentas, we were interested in addressing its possible immunoregulatory effects on T cells. Activated Jurkat T cells and peripheral blood mononuclear cells (PBMCs) were treated with monomeric or dimerized version of a control or a Syn-2 ISD peptide. Change in phosphorylation levels of ERK1/2 MAP kinases was selectively noted in Jurkat cells treated with the dimerized ISD peptide. Upon incubation with the dimerized Syn-2 ISD peptide, significant reduction in Th1 cytokine production was further demonstrated by ELISA and Human Th1/Th2 Panel Multi-Analyte Flow Assay. To determine if exosome-associated Syn-2 could also be immunosuppressive placental exosomes were incubated with activated Jurkat and PBMCs. Quantification of Th1 cytokines in the supernatants revealed severe reduction in T cell activation. Interestingly, exosomes from Syn-2-silenced VCT incubated with PBMCs were less suppressive when compared with exosome derived from VCT transfected with control small interfering RNA (siRNA). Our results suggest that Syn-2 is an important immune regulator both locally and systemically, via its association with placental exosomes.

scholarly journals Involvement of the PD-1/PD-L1 Co-Inhibitory Pathway in the Pathogenesis of the Inflammatory Stage of Early-Onset Preeclampsia

2019 ◽  
Vol 20 (3) ◽  
pp. 583
Author(s):  
Matyas Meggyes ◽  
Eva Miko ◽  
Adrienn Lajko ◽  
Beata Csiszar ◽  
Barbara Sandor ◽  
...  
Keyword(s):  
Natural Killer ◽  
T Cell Activation ◽  
Early Onset ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Cross Sectional ◽  
Effector Cells ◽  
Early Onset Preeclampsia ◽  
In Pregnancy

The programmed cell death protein 1 (PD-1) receptor has been reported to downregulate T cell activation effectively via binding to its ligands PD-L1 or PD-L2 in a negative co-stimulatory manner. Little is known about the involvement of PD-1 mediated immunoregulation in pregnancy and in pregnancy-related disorders. In this work, we investigated the possible role of the PD-1 co-stimulatory pathway in the pathogenesis of the clinical phase of early-onset preeclampsia characterized by a systemic maternal inflammatory response. We performed a cross-sectional study for comparative analysis of phenotypic and functional characteristics of peripheral blood mononuclear cells in women with early-onset preeclampsia and third-trimester healthy pregnant controls. According to our findings, enhanced expression of either PD-1 or its ligand PD-L1, or both, on the cell surface of effector cells (T cells, natural killer (NK) cells, natural killer T (NKT)-like cells) and Tregs could be observed, but PD-1 expression did not correlate with effector cells exhaustion. These results suggest the failure of the axis to downregulate Th1 responses, contributing thereby to the exaggerated immunoactivation observed in early-onset preeclampsia.

scholarly journals DNAM1 and TIGIT balance the T cell response, with low T cell TIGIT expression corresponding to inflammation in psoriatic disease

2020 ◽  
Author(s):  
M E Jacobs ◽  
J N Pouw ◽  
M A Olde Nordkamp ◽  
T R D J Radstake ◽  
E F A Leijten ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Cytokine Production ◽  
Inverse Correlation ◽  
T Cell Response ◽  
Peripheral Blood Mononuclear ◽  
Psoriatic Disease

Abstract Background Signals at the contact site of antigen-presenting cells (APCs) and T cells help orchestrate the adaptive immune response. CD155 on APCs can interact with the stimulatory receptor DNAM1 or inhibitory receptor TIGIT on T cells. The CD155/DNAM1/TIGIT axis is under extensive investigation as immunotherapy target in inflammatory diseases including cancer, chronic infection and autoimmune diseases. We investigated a possible role for CD155/DNAM1/TIGIT signaling in psoriatic disease. Methods By flow cytometry we analyzed peripheral blood mononuclear cells of patients with psoriasis (n=20) or psoriatic arthritis (n=21), and healthy individuals (n=7). We measured CD155, TIGIT and DNAM1 expression on leukocyte subsets and compared activation-induced cytokine production between CD155-positive and -negative APCs. We assessed the effects of TIGIT and DNAM1 blockade on T cell activation, and related the expression of CD155/DNAM1/TIGIT axis molecules to measures of disease activity. Results High CD155 expression associates with TNF production in myeloid and plasmacytoid dendritic cells (DC). In CD1c+ myeloid DC, activation-induced CD155 expression associates with increased HLA-DR expression. CD8 T cells - but not CD4 T cells - express high levels of TIGIT. DNAM1 blockade decreases T cell pro-inflammatory cytokine production, while TIGIT blockade increased T cell proliferation. Finally, T cell TIGIT expression shows an inverse correlation with inflammation biomarkers in psoriatic disease. Conclusion CD155 is increased on pro-inflammatory APCs, while the receptors DNAM1 and TIGIT expressed on T cells balance the inflammatory response by T cells. In psoriatic disease, low TIGIT expression on T cells is associated with systemic inflammation.

scholarly journals Characterization of the T-Cell Receptor Vβ Repertoire in the Human Immune Response against Leishmania Parasites

2006 ◽  
Vol 74 (8) ◽  
pp. 4757-4765 ◽  
Author(s):  
Jorge Clarêncio ◽  
Camila I. de Oliveira ◽  
Glória Bomfim ◽  
Margarida M. Pompeu ◽  
Maria Jania Teixeira ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Cell Receptor ◽  
Peripheral Blood Mononuclear ◽  
Cell Clones ◽  
Human Immune Response ◽  
Lymph Node Cells

ABSTRACT In order to explore a possible presence of hyperreactive T-cell clones in human cutaneous leishmaniasis (CL), we have investigated, by flow cytometry, the expression of Vβ chains of T-cell receptors (TCRs) in the following types of cells: (i) peripheral blood mononuclear cells (PBMCs) from CL patients, which were then compared to those from normal volunteers; (ii) unstimulated and soluble Leishmania antigen-stimulated draining lymph node cells from CL patients; (iii) PBMCs from volunteers before versus after Leishmania immunization; and (iv) PBMCs from healthy volunteers that were primed in vitro with live Leishmania parasites. Our results show a modulation in the TCR Vβ repertoire during CL and after antigen stimulation of patients' cells. Vaccination, however, leads to a broad expansion of different Vβ TCRs. We also observed an association between TCR Vβ12 expression, T-cell activation, and gamma interferon production upon in vitro priming with Leishmania. Collectively, these results both indicate that infection with live parasites or exposure to parasite antigen can modulate the TCR Vβ repertoire and suggest that TCR Vβ12 may be implicated in the response to Leishmania.

scholarly journals Differential effects of IL-21 and IL-15 on perforin expression, lysosomal degranulation, and proliferation in CD8 T cells of patients with human immunodeficiency virus-1 (HIV)

Blood ◽  
2006 ◽  
Vol 109 (9) ◽  
pp. 3873-3880 ◽  
Author(s):  
Lesley White ◽  
Subramaniam Krishnan ◽  
Natasa Strbo ◽  
Huanliang Liu ◽  
Michael A. Kolber ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Highly Active Antiretroviral Therapy ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Cell Receptor ◽  
Peripheral Blood Mononuclear ◽  
Perforin Expression

Abstract An urgent need exists to devise strategies to augment antiviral immune responses in patients with HIV who are virologically well controlled and immunologically stable on highly active antiretroviral therapy (HAART). The objective of this study was to compare the immunomodulatory effects of the cytokines interleukin (IL)–21 with IL-15 on CD8 T cells in patients with HIV RNA of less than 50 copies/mL and CD4 counts greater than 200 cells/mm.3 Patient CD8 T cells displayed skewed maturation and decreased perforin expression compared with healthy controls. Culture of freshly isolated patient peripheral-blood mononuclear cells (PBMCs) for 5 hours to 5 days with IL-21 resulted in up-regulation of perforin in CD8 T cells, including memory and effector subsets and virus-specific T cells. IL-21 did not induce T-cell activation or proliferation, nor did it augment T-cell receptor (TCR)–induced degranulation. Treatment of patient PBMCs with IL-15 resulted in induction of perforin in association with lymphocyte proliferation and augmentation of TCR-induced degranulation. Patient CD8 T cells were more responsive to cytokine effects than the cells of healthy volunteers. We conclude that CD8 T cells of patients with HIV can be modulated by IL-21 to increase perforin expression without undergoing overt cellular activation. IL-21 could potentially be useful for its perforin-enhancing properties in anti-HIV immunotherapy.

Reproducibility and Robustness of the Xcellerate III Process for the GMP Manufacture of Xcellerated T Cells™ for Infusion into CLL Patients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4995-4995
Author(s):  
Lisa Hami ◽  
Cherie Green ◽  
Katharine Miller ◽  
Stewart Craig
Keyword(s):  
T Cells ◽  
Cell Viability ◽  
T Cell Activation ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Total Cell ◽  
Cell Yield ◽  
P Value ◽  
Peripheral Blood Mononuclear ◽  
Significant Difference

Abstract Autologous peripheral blood mononuclear cells (PBMC) cryopreserved from a leukapheresis collection comprise the starting cellular source for the Wave® Bioreactor-based Xcellerate III Process [Hami et al, Bioprocessing Journal2003: 2; 23–32] used for the GMP manufacture of Xcellerated T Cells. For an ongoing clinical trial, n=13 patients have been infused with products manufactured from PBMC cryopreserved and stored in the vapor phase of liquid nitrogen (LN2) for 2–9 days before use in the Xcellerate III Process. This clinical protocol was recently amended to allow patients to receive a 2nd infusion of Xcellerated T Cells. To date, 2nd products have been manufactured for 11 of the CLL patients using the original PBMC that had been stored cryopreserved for up to 7 months from collection. Comparison of the processes for the manufacture of 1st (n=13) and 2nd (n=11) infusion products shows: • No significant difference in the in-process T cell activation as determined by increase in cell size, up-regulation of CD25 & up-regulation of CD154 expression (refer to Figure 1). Figure Figure • The total cell yield for 2nd infusion products is within one (1) standard deviation of the average for the manufacture of the 1st infusion product (refer to Table 1). • No significant difference in cell viability, CD3+ purity, or CD4:CD8 ratio for the final Xcellerated T Cells product (refer to Table 1). Table 1. Final Product Characteristics Final Product (Day 13) Average±S.D. p value 1st Infusion (n=13) 2nd Infusion (n=11) Total Cell Yield (x109) 0.01 137 ± 35 104 ± 17 Cell Viability (%) 0.15 93.5 ± 3.4 91.6 ± 2.4 CD3+ Purity (%) <0.001 98.4 ± 1.1 99.0 ± 0 CD4:CD8 Ratio 0.32 8.5 ± 7.7 5.7 ± 4.5 These data demonstrate high reproducibility and robustness of the Xcellerate III Process when using PBMC from the same leukapheresis collection in sequential processing runs. In addition, these data demonstrate that cryopreserved PBMC can be stored for many months prior to their use as the starting material in the Xcellerate III Process. Xcyte™, Xcyte Therapies™, Xcellerate™, Xcellerated T Cells™ and the circle logo are trademarks of Xcyte Therapies, Inc.

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