In vitro methods for generating highly purified EBV associated tumor antigen-specific T cells by using solid phase T cell selection system for immunotherapy

Nephropathy associated with BK virus has emerged as an important cause of allograft failure in renal transplant recipients. Here we exploited a recently developed novel monocyte based solid phase T cell selection system, in which monocytes are immobilized on solid support, for antigen-specific T cell purification. The underlying hypothesis of this new method is that antigen-specific T cells recognize, bind their cognate antigens faster than non-specific T cells and are concentrated on the surface after removing the non-adherent cells by washing. Moreover, activated antigen-specific T cells proliferate more rapidly than non-specific T cells, further increasing the frequency and purity of antigen-specific T cells. Optimal selection times for BK virus-specific T cells are studied. Our data demonstrated that T cell selection can usually increase the frequency of antigen-specific T cells by > 10 fold, whereas T cell expansion following the selection boost the frequency of antigen-specific T cells by another ~10 fold. This new T cell selection system is superior to traditional stimulation method (i.e. simply mixing antigen presenting cells and lymphocytes together) in generating antigen-specific T cells. This inexpensive and simple T cell selection system can produce large quantity of highly purified BK virus-specific T cells within 1–2 weeks after initial T cell activation.

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Purification of Melanoma Reactive T Cell by Using a Monocyte-based Solid Phase T-cell Selection System for Adoptive Therapy

Journal of Immunotherapy ◽

10.1097/cji.0b013e318157c668 ◽

2008 ◽

Vol 31 (1) ◽

pp. 81-88 ◽

Cited By ~ 6

Author(s):

Jongming Li ◽

Bijoyesh Mookerjee ◽

John Wagner

Keyword(s):

T Cell ◽

Solid Phase ◽

Cell Selection ◽

Selection System ◽

Adoptive Therapy ◽

T Cell Selection

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CD4/CD8 T-Cell Selection Affects Chimeric Antigen Receptor (CAR) T-Cell Potency and Toxicity: Updated Results From a Phase I Anti-CD22 CAR T-Cell Trial

Journal of Clinical Oncology ◽

10.1200/jco.19.03279 ◽

2020 ◽

Vol 38 (17) ◽

pp. 1938-1950 ◽

Cited By ~ 24

Author(s):

Nirali N. Shah ◽

Steven L. Highfill ◽

Haneen Shalabi ◽

Bonnie Yates ◽

Jianjian Jin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Phase I ◽

Chimeric Antigen Receptor ◽

Cd8 T Cell ◽

Cell Selection ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

T Cell Selection

PURPOSE Patients with B-cell acute lymphoblastic leukemia who experience relapse after or are resistant to CD19-targeted immunotherapies have limited treatment options. Targeting CD22, an alternative B-cell antigen, represents an alternate strategy. We report outcomes on the largest patient cohort treated with CD22 chimeric antigen receptor (CAR) T cells. PATIENTS AND METHODS We conducted a single-center, phase I, 3 + 3 dose-escalation trial with a large expansion cohort that tested CD22-targeted CAR T cells for children and young adults with relapsed/refractory CD22+ malignancies. Primary objectives were to assess the safety, toxicity, and feasibility. Secondary objectives included efficacy, CD22 CAR T-cell persistence, and cytokine profiling. RESULTS Fifty-eight participants were infused; 51 (87.9%) after prior CD19-targeted therapy. Cytokine release syndrome occurred in 50 participants (86.2%) and was grade 1-2 in 45 (90%). Symptoms of neurotoxicity were minimal and transient. Hemophagocytic lymphohistiocytosis–like manifestations were seen in 19/58 (32.8%) of subjects, prompting utilization of anakinra. CD4/CD8 T-cell selection of the apheresis product improved CAR T-cell manufacturing feasibility as well as heightened inflammatory toxicities, leading to dose de-escalation. The complete remission rate was 70%. The median overall survival was 13.4 months (95% CI, 7.7 to 20.3 months). Among those who achieved a complete response, the median relapse-free survival was 6.0 months (95% CI, 4.1 to 6.5 months). Thirteen participants proceeded to stem-cell transplantation. CONCLUSION In the largest experience of CD22 CAR T-cells to our knowledge, we provide novel information on the impact of manufacturing changes on clinical outcomes and report on unique CD22 CAR T-cell toxicities and toxicity mitigation strategies. The remission induction rate supports further development of CD22 CAR T cells as a therapeutic option in patients resistant to CD19-targeted immunotherapy.

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Cathepsin L Regulates CD4+ T Cell Selection Independently of Its Effect on Invariant Chain

Journal of Experimental Medicine ◽

10.1084/jem.20011904 ◽

2002 ◽

Vol 195 (10) ◽

pp. 1349-1358 ◽

Cited By ~ 124

Author(s):

Karen Honey ◽

Terry Nakagawa ◽

Christoph Peters ◽

Alexander Rudensky

Keyword(s):

T Cells ◽

T Cell ◽

Epithelial Cells ◽

Positive Selection ◽

Cathepsin L ◽

Cd4 T Cell ◽

Thymic Epithelial Cells ◽

Invariant Chain ◽

Cell Selection ◽

T Cell Selection

CD4+ T cells are positively selected in the thymus on peptides presented in the context of major histocompatibility complex class II molecules expressed on cortical thymic epithelial cells. Molecules regulating this peptide presentation play a role in determining the outcome of positive selection. Cathepsin L mediates invariant chain processing in cortical thymic epithelial cells, and animals of the I-Ab haplotype deficient in this enzyme exhibit impaired CD4+ T cell selection. To determine whether the selection defect is due solely to the block in invariant chain cleavage we analyzed cathepsin L–deficient mice expressing the I-Aq haplotype which has little dependence upon invariant chain processing for peptide presentation. Our data indicate the cathepsin L defect in CD4+ T cell selection is haplotype independent, and thus imply it is independent of invariant chain degradation. This was confirmed by analysis of I-Ab mice deficient in both cathepsin L and invariant chain. We show that the defect in positive selection in the cathepsin L−/− thymus is specific for CD4+ T cells that can be selected in a wild-type and provide evidence that the repertoire of T cells selected differs from that in wild-type mice, suggesting cortical thymic epithelial cells in cathepsin L knockout mice express an altered peptide repertoire. Thus, we propose a novel role for cathepsin L in regulating positive selection by generating the major histocompatibility complex class II bound peptide ligands presented by cortical thymic epithelial cells.

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Functional and Molecular Analysis of Maturation of Thymocytes in Bcl10 or Malt1 Deficient Mice.

Blood ◽

10.1182/blood.v106.11.3323.3323 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3323-3323

Author(s):

Philipp J. Jost ◽

Uta Ferch ◽

Stephanie Weiss ◽

Stephanie Leeder ◽

Olaf Gross ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Transgenic Animals ◽

Adaptor Proteins ◽

Cell Receptor ◽

Single Positive ◽

T Cell Selection ◽

Deficient Mice

Abstract Development of immature T cells in the thymus requires signals through the clonotypic T cell receptor (TCR). Thymocytes expressing a functionally inactive or autoreactive TCR are deleted via apoptosis (negative selection). Thymocytes expressing a functionally active but not autoreactive TCR are selected through inhibition of cell death (positive selection). Deregulation of this process is likely to result in autoimmunity or lymphomagenesis of T cells. The intracellular mechanisms by which the balance between TCR-dependent survival and apoptosis are regulated are largely unknown. A central regulator of survival and apoptosis in the immune system is the transcription factor NF-κB. Activation of NF-κB in mature T-cells requires the adaptor proteins Bcl10 and Malt1. Using gene-targeted mice deficient for Bcl10 or Malt1, we show that Bcl10 and Malt1 are also required for TCR-induced NF-κB activation in immature T cells. Furthermore, to elucidate the process of T cell selection within the thymus, we have crossed Bcl10 or Malt1 deficient mice into mice with genetic backgrounds expressing defined TCR transgenes. Using specific peptide agonists of these TCR transgenes, we show that neither in vivo nor in vitro development into single positive (SP) CD4 or CD8 positive T cells is altered in Bcl10 or Malt1 deficient mice. Absolute numbers and ratio of SP T cells found within the thymus or in peripheral lymphnodes of transgenic animals are normal. These findings indicate that Bcl10 and Malt1 activate NF-κB in thymocytes but are dispensable for maturation of immature T cells in this model system.

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T-Cell Immune Reconstitution in Pediatric Leukemia Patients After Allogeneic Bone Marrow Transplantation With T-Cell–Depleted or Unmanipulated Grafts: Evaluation of Overall and Antigen-Specific T-Cell Repertoires

Blood ◽

10.1182/blood.v94.12.4358.424k02_4358_4369 ◽

1999 ◽

Vol 94 (12) ◽

pp. 4358-4369 ◽

Cited By ~ 2

Author(s):

Barbara C. Godthelp ◽

Maarten J.D. van Tol ◽

Jaak M. Vossen ◽

Peter J. van den Elsen

Keyword(s):

Bone Marrow ◽

T Cells ◽

Bone Marrow Transplantation ◽

T Cell ◽

Marrow Transplantation ◽

Immune Reconstitution ◽

Allogeneic Bone Marrow Transplantation ◽

Cell Selection ◽

Allogeneic Bone ◽

T Cell Selection

To evaluate the role of T-cell selection in the thymus and/or periphery in T-cell immune reconstitution after allogeneic bone marrow transplantation (allo-BMT), we have analyzed the overall and antigen-specific T-cell repertoires in pediatric allo-BMT recipients treated for leukemia. We observed a lack of overall T-cell receptor (TCR) diversity in the repopulating T cells at 3 months after allo-BMT, as was deduced from complementarity determining region 3 (CDR3) size distribution patterns displaying reduced complexity. This was noted particularly in recipients of a T-cell–depleted (TCD) graft and, to a lesser extent, also in recipients of unmanipulated grafts. At 1 year after allo-BMT, normalization was observed of TCR CDR3 size complexity in almost all recipients. Analysis of the antigen-specific T-cell repertoire at 1 year after BMT showed that the T cells responding to tetanus toxoid (TT) differed in TCR gene segment usage and in amino acid composition of the CDR3 region when comparing the recipient with the donor. Moreover, the TT-specific TCR repertoire was found to be stable within a given allo-BMT recipient, because TT-specific T cells with completely identical TCRs were found at 3 consecutive years after transplantation. These observations suggest an important role for T-cell selection processes in the complete restoration of the T-cell immune repertoire in children after allo-BMT.

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Tumor Antigen-Dependent and Tumor Antigen-Independent Activation of Antitumor Activity in T Cells by a Bispecific Antibody-Modified Tumor Vaccine

Clinical and Developmental Immunology ◽

10.1155/2010/423781 ◽

2010 ◽

Vol 2010 ◽

pp. 1-12 ◽

Cited By ~ 2

Author(s):

Philippe Fournier ◽

Volker Schirrmacher

Keyword(s):

T Cells ◽

T Cell ◽

Antitumor Activity ◽

Cancer Patients ◽

Tumor Antigen ◽

Immune Escape ◽

Tumor Vaccine ◽

Cell Repertoire ◽

Tumor Immune Escape

New approaches of therapeutic cancer vaccination are needed to improve the antitumor activity of T cells from cancer patients. We studied over the last years the activation of human T cells for tumor attack. To this end, we combined the personalized therapeutic tumor vaccine ATV-NDV—which is obtained by isolation, shortin vitroculture, irradiation, and infection of patient's tumor cells by Newcastle Disease Virus (NDV)—with bispecific antibodies (bsAbs) binding to this vaccine and introducing anti-CD3 (signal 1) and anti-CD28 (signal 2) antibody activities. This vaccine called ATV-NDV/bsAb showed the unique ability to reactivate a preexisting potentially anergized antitumor memory T cell repertoire. But it also activated naive T cells to have antitumor propertiesin vitroandin vivo. This innovative concept of direct activation of cancer patients' T cells via cognate and noncognate interactions provides potential for inducing strong antitumor activities aiming at overriding T cell anergy and tumor immune escape mechanisms.

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The CCR4–NOT deadenylase complex safeguards thymic positive selection by down-regulating aberrant pro-apoptotic gene expression

Nature Communications ◽

10.1038/s41467-020-19975-4 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Taku Ito-Kureha ◽

Takahisa Miyao ◽

Saori Nishijima ◽

Toru Suzuki ◽

Shin-ichi Koizumi ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

Positive Selection ◽

Early Stage ◽

Cell Selection ◽

Antigen Receptors ◽

Apoptotic Gene ◽

Thymocyte Apoptosis ◽

T Cell Selection

AbstractA repertoire of T cells with diverse antigen receptors is selected in the thymus. However, detailed mechanisms underlying this thymic positive selection are not clear. Here we show that the CCR4-NOT complex limits expression of specific genes through deadenylation of mRNA poly(A) tails, enabling positive selection. Specifically, the CCR4-NOT complex is up-regulated in thymocytes before initiation of positive selection, where in turn, it inhibits up-regulation of pro-apoptotic Bbc3 and Dab2ip. Elimination of the CCR4-NOT complex permits up-regulation of Bbc3 during a later stage of positive selection, inducing thymocyte apoptosis. In addition, CCR4-NOT elimination up-regulates Dab2ip at an early stage of positive selection. Thus, CCR4-NOT might control thymocyte survival during two-distinct stages of positive selection by suppressing expression levels of pro-apoptotic molecules. Taken together, we propose a link between CCR4-NOT-mediated mRNA decay and T cell selection in the thymus.

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T-cells with a single tumor antigen-specific T-cell receptor can be generated in vitro from clinically relevant stem cell sources

OncoImmunology ◽

10.1080/2162402x.2020.1727078 ◽

2020 ◽

Vol 9 (1) ◽

pp. 1727078 ◽

Cited By ~ 1

Author(s):

Sarah Bonte ◽

Stijn De Munter ◽

Glenn Goetgeluk ◽

Joline Ingels ◽

Melissa Pille ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

T Cell Receptor ◽

Tumor Antigen ◽

Cell Receptor ◽

Cell Sources ◽

Single Tumor

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SELECTIVE INHIBITION OF HUMAN MEGAKARYOCYTOPOIESIS IN VITRO BY HIGHLY PURIFIED PLATELET FACTOR 4

10.1055/s-0038-1644621 ◽

1987 ◽

Author(s):

A Gewirtz ◽

W Y Xu ◽

B Rucinski ◽

S Niewiarowski

Keyword(s):

T Cells ◽

T Cell ◽

Colony Formation ◽

Solid Phase ◽

Specific Inhibitor ◽

Platelet Glycoprotein ◽

Lymphocyte Function ◽

Erythroid Progenitor ◽

Platelet Factor

Platelet (plt) factor 4 (PF4) is an alpha granule protein which can modulate T lymphocyte function. T cells may help regulate megakaryocytopoiesis. Therefore, we hypothesized that T cell-PF4 interactions might play a role in autoregulating marrow megakaryocyte (MEG) production. To test this idea, we studied MEG colony formation in plasma clot cultures containing human serum derived solely from pit poor normal AB plasma, enriched hematopoietic progenitor cells (HPC), autologous T cells, and exogenous PF4. Highly purified PF4 (single band on SDS gel) was prepared from outdated human pits by a combination of heparin-agarose, Sephacryl G-200, and Sephadex G-50 column chromatography. HPC were prepared by depleting normal light density marrow mononuclear cells of adherent monocytes, and T cells. T cells were further fractionated into helper (Leu 3+) and suppressor (Leu 2+) subtypes by solid phase immunoabsorption ("panning"). MEG colonies were enumerated by indirect immunofluorescence with an anti-human platelet glycoprotein antiserum. HPC(5×105/ml) were co-cultured with Leu 3+, or Leu 2+ T cells at target;T cell ratios of 2:1 (n=3; n=4 respectively) and l:l(n=4; n=4 respectively) in the presence of 2.5 μg/ml PF4. Under these growth conditions, MEG colony formation was unchanged (p>0.5) when compared to colonies formed by HPC in the absence of PF4. When the above experiments were repeated (n=2-3/condition) at a higher PF4 concentration [25 μg/ml], MEG colony formation was markedly (>60%) inhibited. To determine if PF4 directly inhibited MEG or erythroid progenitor cell growth (CFU-Meg; CFU-E) in vitro, HPC were cloned in PF4 (25μg/ml) without added T cells. Mean ± SEM of MEG and CFU-E derived colonies formed without vs. with PF4 was as follows:These results suggest that: 1) PF4 may be a non-T cell dependent, lineage specific inhibitor of CFU-MEG, and 2) PF4 may play a role in autoregulating human megakaryocytopoiesis.

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