In Vitro Stimulation and Expansion of Human Tumour-Reactive CD8+ Cytotoxic T Lymphocytes by Anti-CD3/CD28/CD137 Magnetic Beads

Adoptive immunotherapy with tumour-reactive CD8(+) cytotoxic T lymphocytes (CTLs) requires efficient in vitro approaches allowing the expansion of CTLs to large numbers prior infusion. Here, we investigated the antigen-independent activation and the expansion of human T cells in peripheral blood mononuclear cells (PBMCs) and in tumour-reactive CTLs using Dynabeads coated with monoclonal antibodies to CD3 and to the costimulatory molecules CD28 and CD137 (4-1BB). T cells in PBMCs showed an increased expansion rate of 15- to 17-fold during a 2-week culture period using antibody-conjugated beads with interleukin-2 (IL-2) added versus IL-2 alone. No significant difference between CD3/CD28 beads…

The CD38-Positive and CD38-Negative Subsets of CD34(high)-Positive Primary Acute Myeloid Leukemia Blasts Differ Considerably in the Expression of Immune Recognition Molecules

Abstract Acute myeloid leukemia (AML) is thought to arise from a rare putative ‘leukemic stem cell’ that is capable of self-renewal and formation of leukemic blasts. Serial xenotransplantation studies in immunodeficient mice have shown that this leukemia-initiating cell resides at very low numbers within CD34(high)-positive CD38-negative AML cells. Thus, immunotherapeutic approaches successfully eradicating this cell compartment should result in cure from disease. The objective of our study was to characterize the immune phenotype of the CD38-negative and CD38-positive subsets of primary CD34(high)-positive AML blasts ex vivo. We obtained therapeutic leukapheresis products from 17 AML patie…

HLA-DPB1 mismatch alleles represent powerful leukemia rejection antigens in CD4 T-cell immunotherapy after allogeneic stem-cell transplantation.

Refractory or relapsed acute myeloid leukemia (AML) represents a frequent complication after allogeneic hematopoietic stem-cell transplantation (HSCT). We show herein that primary in vitro stimulation of CD45RA-selected CD4 T cells of stem-cell donors with 10/10 HLA-matched AML blasts results in expansion of cytolytic T-lymphocytes (CTL) that almost all recognize HLA-DPB1 mismatch alleles, which clinically occur in up to 80% of donor-patient pairs. Primary AML blasts were found to strongly express HLA-DPB1, whereas fibroblasts and keratinocytes used as surrogate target cells for graft-versus-host disease did express HLA-DPB1 only upon IFN-γ pre-treatment. Since patients' AML blasts are rare…

Alloreactive and leukemia-reactive T cells are preferentially derived from naive precursors in healthy donors: implications for immunotherapy with memory T cells.

Background HLA mismatch antigens are major targets of alloreactive T cells in HLA-incompatible stem-cell transplantation, which can trigger severe graft- versus -host disease and reduce survival in transplant recipients. Our objective was to identify T-cell subsets with reduced in vitro reactivity to allogeneic HLA antigens. Design and Methods We sorted CD4 and CD8 T-cell subsets from peripheral blood by flow cytometry according to their expression of naive and memory markers CD45RA, CD45RO, CD62L, and CCR7. Subsets were defined by a single marker to facilitate future establishment of a clinical-grade procedure for reducing alloreactive T-cell precursors and graft- versus -host disease. T c…

Preservation of dendritic cell function upon labeling with amino functionalized polymeric nanoparticles.

Dendritic cells (DCs) are key players in eliciting immunity against antigens, therefore making them the focus of many investigations on immune responses in infections, cancer and autoimmune diseases. Nanosized materials have just recently been investigated for their use as carriers of antigens and as labeling agents for DCs. For this later use nanoparticles should be non-toxic and should most importantly not alter the physiological functions of DCs. Here we demonstrate that by the use of polymeric fluorescent nanoparticles as synthesized by the miniemulsion process immature DCs (iDCs) can be efficiently labeled intracellularly. Amino functionalized nanoparticles are more effective than carb…

29: Rapid expansion of acute myeloid leukemia-reactive cytotoxic T cells from CD8+CD62L+ blood lymphocytes of HLA-matched healthy donors in vitro

Nanoparticles and antigen-specific T-cell therapeutics: A comprehensive study on uptake and release

Aim: T lymphocytes are used as cellular therapeutics in many disease entities including cancer. We investigated the uptake and retention of nanoparticles (NPs) by these nonphagocytic cells. Materials & methods: Uptake, release and toxicity of various polymeric NP preparations were analyzed by flow cytometry, confocal laser scanning microscopy and transmission electron microscopy. T-cell effector functions were measured using IFN-γ-ELISPOT and 51Chromium-release assays. Results: Amino-functionalized NPs were efficiently ingested by antigen-specific T cells without adversely influencing effector functions. NPs were stored in membrane-surrounded vesicles, with major proportions released e…

Selective Depletion of Alloreactive T Lymphocytes Using Patient-Derived Nonhematopoietic Stimulator Cells in Allograft Engineering

Background. Selective depletion of alloreactive T cells in vitro results in efficient graft-versus-host disease prophylaxis in allogeneic hematopoietic stem-cell transplantation, but it is accompanied by increased recurrence of leukemia. To spare donor T-cell-mediated graft-versus-leukemia immunity against hematopoiesis-restricted minor histocompatibility (minor-H) antigens, we explored the use of patient-derived nonhematopoietic antigen-presenting cells (APC) as allogeneic stimulators for selective allodepletion in leukemia-reactive donor T-cell lines. Methods. Primary keratinocytes, dermal fibroblasts, and bone marrow fibroblasts were generated from skin biopsies and diagnostic bone marro…

Patient-individualized CD8+cytolytic T-cell therapy effectively combats minimal residual leukemia in immunodeficient mice

Adoptive transfer of donor-derived cytolytic T-lymphocytes (CTL) has evolved as a promising strategy to improve graft-versus-leukemia (GvL) effects in allogeneic hematopoietic stem-cell transplantation. However, durable clinical responses are often hampered by limited capability of transferred T cells to establish effective and sustained antitumor immunity in vivo. We therefore analyzed GvL responses of acute myeloid leukemia (AML)-reactive CD8(+) CTL with central and effector memory phenotype in a new allogeneic donor-patient specific humanized mouse model. CTL lines and clones obtained upon stimulation of naive CD45RA(+) donor CD8(+) T cells with either single HLA antigen-mismatched or HL…

In Vitro Expansion of Human Tumor-Reactive CD8+ T Cell Lines Using Superparamagnetic CD3/CD28/CD137 Beads.

Abstract Abstract 5118 Introduction Efficient methods for the reliable in vitro expansion of tumor-reactive T cells will surely broaden the applicability of adoptive T cell therapy in cancer. In this study we investigated the antigen-independent stimulation and expansion of human T cells in peripheral blood mononuclear cells (PBMC) and in long-term cultured tumor-reactive CD8+ T cell lines using superparamagnetic beads coated with antibodies to CD3 and the costimulatory molecules CD28 and CD137. Methods T cell numbers were measured in healthy donor PBMC after in vitro stimulation with Dynabeads® coated with CD3/CD28/CD137 versus Dynabeads® coated with CD3/CD28 (all beads +/- 100 U/mL IL-2) …

Rapid Expansion of Acute Myeloid Leukemia-Reactive Cytotoxic T Cells from CD8+CD62L+ Blood Lymphocytes of HLA-Matched Healthy Donors In Vitro

Abstract Allogeneic cytotoxic T-lymphocyte (CTL) therapy in acute myeloid leukemia (AML) is hampered by the poor efficiency of growing leukemia-reactive CTLs from healthy donors in vitro. We established an allogeneic mini-mixed lymphocyte-leukemia culture (MLLC) approach by stimulating comparably small numbers (104/well) of CD8+ T cells isolated from healthy donors against irradiated primary AML blasts in 96-well plates. Prior to use, CD8+ T cells were immunomagnetically separated into a CD62L(high)+ subset enriched for naive precursors and central memory cells as well as a CD62L(low)+/negative subset containing effector memory cells. The culture medium contained IL-7, IL-12, and IL-15. Aft…

Acute myeloid leukemia (AML)-reactive cytotoxic T lymphocyte clones rapidly expanded from CD8(+) CD62L((high)+) T cells of healthy donors prevent AML engraftment in NOD/SCID IL2Rgamma(null) mice.

Objective Current in vitro techniques for isolating leukemia-reactive cytotoxic T lymphocytes (CTLs) from healthy donors are of relatively low efficiency and yield responder populations with unknown biological significance. This study aimed at the development of a more reliable approach, allowing generation and expansion of acute myeloid leukemia (AML)-reactive CTLs using primary in vitro stimulation. Materials and Methods We established allogeneic mini-mixed lymphocyte-leukemia cultures (mini-MLLCs) by stimulating donor CD8 + T cells with human leukocyte antigen (HLA) class I–matched AML blasts in microtiter plates. Before culture, CD8 + T cells were separated into CD62L (high)+ and CD62L …

Rapid identification and sorting of viable virus-reactive CD4+ and CD8+ T cells based on antigen-triggered CD137 expression

Abstract Current methods for the detection and isolation of antigen-specific CD4 + and CD8 + T cells require the availability of peptide/MHC multimers or are restricted to cells that produce cytokines after antigen contact. Here we show that de novo cell surface expression of the TNF-receptor family member CD137 (4-1BB) identifies recently activated, but not resting, human CD4 + and CD8 + memory T cells. Maximum CD137 expression level is uniformly observed in both T-cell subsets at 24h after stimulation with antigen. In experiments with CMV and EBV-reactive T cells, we confirmed the specificity of CD137 expression by co-staining with peptide/HLA tetramers. Substantial proportions of CD137 +…

Recovery of Varicella-Zoster Virus–Specific T Cell Immunity after T Cell–Depleted Allogeneic Transplantation Requires Symptomatic Virus Reactivation

Abstract Reactivated varicella-zoster virus (VZV) infection causes herpes zoster and commonly occurs after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Because VZV-specific T cell immunity is essential to prevent virus reactivation, we developed an interferon-γ enzyme-linked immunosorbent spot (ELISPOT) assay for the sensitive detection of VZV-reactive T cells at the single-cell level ex vivo. We used this assay to monitor the frequency of VZV-reactive T cells in 17 seropositive patients during the first year after T cell–depleted allo-HSCT. The patients did not receive anti-herpesvirus prophylaxis after stem cell engraftment. Independent of the magnitude of transferred d…

Combining dasatinib with dexamethasone long-term leads to maintenance of antiviral and antileukemia specific cytotoxic T cell responses in vitro

Maintaining graft versus leukemia (GvL) and antivirus responses of cytotoxic T cells (CTLs) while suppressing graft-versus-host disease (GvHD) remains a challenge after allogeneic bone marrow transplantation. Clinical observations indicate that combining glucocorticoids with multi-tyrosine-kinase inhibitors could be a successful therapeutic approach. We and others have shown that the BCR-ABL/SRC kinase inhibitor dasatinib may enhance or suppress T cells in vitro. In this report, we evaluated combination effects of dasatinib and dexamethasone on CD3 + and virus-specific CD8 + T cells directly ex vivo and on antigen-specific leukemia-reactive and alloreactive CD8 + T cell clones. Functional o…

The Programmed Death (PD)‐1/PD‐Ligand 1 Pathway Regulates Graft‐Versus‐Host‐Reactive CD8 T Cells After Liver Transplantation

Acute graft-versus-host disease (aGVHD) is a life-threatening complication after solid-organ transplantation, which is mediated by host-reactive donor T cells emigrating from the allograft. We report on two liver transplant recipients who developed an almost complete donor chimerism in peripheral blood and bone marrow-infiltrating T cells during aGVHD. By analyzing these T cells directly ex vivo, we found that they died by apoptosis over time without evidence of rejection by host T cells. The host-versus-donor reactivity was selectively impaired, as anti-third-party and antiviral T cells were still detectable in the host repertoire. These findings support the acquired donor-specific allotol…

Depletion of naive T cells using clinical grade magnetic CD45RA beads: a new approach for GVHD prophylaxis

Depletion of naive T cells from donor leukapheresis products (LPs) aims at the reduction of alloreactivity, while preserving memory T-cell reactivity (for example, to pathogens). This study established the immunomagnetic depletion procedure under clean room conditions using CD45RA beads and analyzed LPs of six donors for cell composition and functional immune responses. CD45RA depletion resulted in 3.4-4.7 log (median 4.4) reduction of CD45RA(+) T cells, thereby eliminating naive and late effector T cells. B cells were also completely removed, whereas significant proportions of NK cells, monocytes and granulocytes persisted. CD45RA-depleted LPs contained effector and central memory CD4(+) a…

Graft-Versus-Host Disease or Infection: Rapid Detection of HLA Mismatch-Reactive T Cells Ex Vivo Can Facilitate Diagnosis and Guide Therapy after Allogeneic Transplantation.

Abstract Diagnosis of graft-versus-host disease (GVHD) is mainly based on clinical features and on tissue biopsies. However, clinicians and pathologists are well aware of cases, in which GVHD cannot be distinguished from infections arising from severe immunodeficiency after allogeneic stem-cell transplantation (SCT). This may pose a deep therapeutic dilemma of whether to modify immunosuppressive treatment or to use donor lymphocyte infusion (DLI) for promoting anti-microbial immunity. We observed a 68-year-old patient with myelodysplastic syndrome who developed acute GVHD grade II of skin and gut at d+16 after T-cell depleted reduced-intensity SCT (Fig. 1). GVHD was confirmed by histology a…