Autologous Target Cells Research Articles

Expanded Antigen-Specific Elimination Assay to Measure Human CD8+ T Cell Cytolytic Potential.

Durable cellular immunity against pathogens is dependent upon a coordinated recall response to antigen by memory CD8+ T cells, involving their proliferation and the generation of secondary cytotoxic effector cells. Conventional assays measuring ex vivo cytotoxicity fail to capture this secondary cytolytic potential, especially in settings where cells have not been recently exposed to their cognate antigen in vivo. Here we describe the expanded antigen-specific elimination assay (EASEA), a flow cytometric endpoint assay to measure the capacity of human CD8+ T cells to expand in vitro upon antigen re-exposure and generate secondary effector cells capable of selectively eliminating autologous antigen-pulsed target cells across a range of effector-to-target ratios. Unlike alternative assays, EASEA avoids the hazards of radioactive labeling and viral infection and can be used to study responses to individual or pooled antigens of interest. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Expanded antigen-specific elimination assay.

Development of a centrifugal bioreactor for rapid expansion of CD8 cytotoxic T cells for use in cancer immunotherapy.

One of the current difficulties limiting the use of adoptive cell therapy (ACT) for cancer treatment is the lack of methods for rapidly expanding T cells. As described in the present report, we developed a centrifugal bioreactor (CBR) that may resolve this manufacturing bottleneck. The CBR operates in perfusion by balancing centrifugal forces with a continuous feed of fresh medium, preventing cells from leaving the expansion culture chamber while maintaining nutrients for growth. A bovine CD8 cytotoxic T lymphocyte (CTL) cell line specific for an autologous target cell infected with a protozoan parasite, Theileria parva, was used to determine the efficacy of the CBR for ACT purposes. Batch culture experiments were conducted to predict how CTLs respond to environmental changes associated with consumption of nutrients and production of toxic metabolites, such as ammonium and lactate. Data from these studies were used to develop a kinetic growth model, allowing us to predict CTL growth in the CBR and determine the optimal operating parameters. The model predicts the maximum cell density the CBR can sustain is 5.5 × 107 cells/mL in a single 11-mL conical chamber with oxygen being the limiting factor. Experimental results expanding CTLs in the CBR are in 95% agreement with the kinetic model. The prototype CBR described in this report can be used to develop a CBR for use in cancer immunotherapy.

Abstract 2008: The oncogenic fusion protein DNAJB1-PRKACA can be actively targeted by peptide-based immunotherapy in fibrolamellar hepatocellular carcinoma

Abstract Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare tumor disease, which affects children and adolescents without history of primary liver disease. Beside surgical resection established treatment options are lacking for FL-HCC. Recently, the DNAJB1-PRKACA fusion transcript was identified as the oncogenic driver of tumor pathogenesis in 100% of FL-HCC patients. Here, we investigated the role of the DNAJB1-PRKACA fusion protein as a source for immunogenic neoepitopes and showed first immunotherapeutic application of these antigens in a FL-HCC patient.HLA class I- and class II-presented neoantigens derived from the DNAJB1-PRKACA fusion protein were predicted in silico using NetMHCpan 4.1 and SYFPEITHI 1.0, or NetMHCIIpan 4.0, respectively. With this workflow nine binding cores of nine amino acid length for a total of 1290 different HLA class II alleles, as well as 13 HLA class I ligands for the 20 most frequent HLA class I allotypes (European population, iedb.org) were identified. Cellular processing and HLA presentation of DNAJB1-PRKACA-derived peptides was proven by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) of DNAJB1-PRKACA-transduced HCC cell lines. Immunogenicity of DNAJB1-PRKACA-derived peptides was assessed for the HLA class II peptide (PII-1) and the HLA-A*24 peptide (PA*24) by in vitro priming experiments which showed an induction of multifunctional peptide-specific CD4+ and CD8+ T cells, respectively, with expression of CD107a, IFNγ, and TNF upon peptide-pulsing. Furthermore, PA*24-specific T cells showed antigen-specific lysis of autologous peptide-loaded target cells and single-cell next-generation sequencing (10x Genomics) of PA*24-specific CD8+ T cells further enabled the identification of DNAJB1-PRKACA-reactive T cell receptors. Based on these preclinical data we applied a peptide vaccine, consisting of three HLA class I ligands (PA*02, PB*44, and PC*05) and PII-1 spanning the DNAJB1-PRKACA fusion region, to a 15-year old patient with histologically confirmed FL-HCC, who experienced multiple tumor relapses after early liver transplant due to unresectable FL-HCC not responsive to chemotherapy. After two vaccinations in vivo induction of multifunctional CD4+ T cells targeting PII-1 and PB*44 was observed by IFNγ ELISPOT. Single-cell RNA sequencing of vaccine-induced CD4+ T cells revealed distinct gene expression clusters of T cell activation and high TCR clonality. DNAJB1-PRKACA-specific T cells persisted in peripheral blood and were accompanied by relapse free survival of the patient until now, more than one year post vaccination. These findings identified the DNAJB1-PRKACA fusion transcript as novel prime source for broadly applicable neoepitopes and corresponding TCRs and provide first evidence for their application in cancer immunotherapy of FL-HCC. Citation Format: Jens Bauer, Natalie Köhler, Yacine Maringer, Philip Bucher, Tatjana Bilich, Melissa Zwick, Severin Dicks, Annika Nelde, Marissa Dubbelaar, Jonas Scheid, Marcel Wacker, Jonas J. Heitmann, Sarah Schroeder, Jonas Rieth, Monika Denk, Marion Richter, Reinhild Klein, Irina Bonzheim, Julia Luibrand, Ursula Holzer, Martin Ebinger, Ines B. Brecht, Michael Bitzer, Melanie Boerries, Helmut R. Salih, Hans-Georg Rammensee, Stephan Hailfinger, Juliane S. Walz. The oncogenic fusion protein DNAJB1-PRKACA can be actively targeted by peptide-based immunotherapy in fibrolamellar hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2008.

MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

ABSTRACT The major histocompatibility complex-class I chain related proteins A and B (MICA/B) is upregulated because of cellular stress and MICA/B shedding by cancer cells causes escape from NKG2D recognition favoring the emergence of cancers. Cholangiocarcinoma (CCA) is a relatively rare, though increasingly prevalent, primary liver cancer characterized by a late clinical presentation and a dismal prognosis. We explored the NKG2D-MICA/B axis in NK cells from 41 patients with intrahepatic cholangiocarcinoma (iCCA). The MICA/B-specific 7C6 mAb was used for ex vivo antibody-dependent cytotoxicity (ADCC) experiments using circulating, non tumor liver- and tumor-infiltrating NK cells against the HuCCT-1 cell line and patient-derived primary iCCA cells as targets. MICA/B were more expressed in iCCA than in non-tumoral tissue, MICA transcription being higher in moderately-differentiated compared with poorly-differentiated cancer. Serum MICA was elevated in iCCA patients in line with higher expression of ADAM10 and ADAM17 that are responsible for proteolytic release of MICA/B from tumor. Addition of 7C6 significantly boosted peripheral, liver- and tumor-infiltrating-NK cell degranulation and IFNγ production toward MICA/B-expressing established cell lines and autologous iCCA patient target cells. Our data show that anti-MICA/B drives NK cell anti-tumor activity, and provide preclinical evidence in support of 7C6 as a potential immunotherapeutic tool for iCCA.

Evaluating Cyclin A1 As an Immunotherapeutic Target in Acute Myeloid Leukemia (AML)

T cell based immunotherapy targeting leukemia associated antigens (LAAs) such as Wilm's tumor 1 antigen (WT1) has proven both safe and clinically effective, with the induction of durable responses in patients with relapsed AML. However, because of heterogeneous LAA expression among leukemic blasts, targeting a single LAA has led to tumor immune escape and relapse, highlighting the need to identify and simultaneously target an array of tumor-expressed LAAs. To expand the target antigen spectrum we sought to immunologically characterize cyclin A1 (CCNA1), a meiosis-specific cell cycle antigen that is selectively expressed in AML blasts with limited normal tissue expression and which has been implicated as playing a role in leukemogenesis based on preclinical animal models.To assess the suitability of CCNA1 as an immunotherapeutic target we first sought to expand reactive populations ex vivo using an overlapping peptide library (15mers overlapping by 11 amino acids) spanning the entire antigen as a source of stimulation. In 12 donors tested we achieved a mean 5.2±0.65 fold expansion of T cells, of which 11 proved to recognize CCN1 as measured by an IFNg ELISpot [mean 377.3 spot forming cells (SFCs)/1x105 T cells (range 38 to 1188 SFCs) ]. The antigen specific lines were polyclonal (23±4% CD4+ and 64±5% CD8+ T cells) that expressed markers for both central and effector memory (29±7% CD45RO+CD62L+ and 28±6% CD45RO+CD62L-), required for long term in vivo persistence. Importantly both helper (CD4+) and cytotoxic (CD8+) subsets contained CCNA1-directed T cell populations, which secreted multiple cytokines (1.47±0.07% CD4+IFNg+TNFa+ and 9.52±0.25% CD8+IFNg+TNFa+) as measured by intracellular cytokine staining, confirming their polyfunctionality. Moreover, CCNA1-specific T cells were able to selectively kill pepmix-loaded autologous target cells and a partially HLA-matched cell line that endogenously-expresses cyclin A1 (44±7% and 55±1%, respectively; E:T 20:1). without non-specific activity against control (unloaded) targets (9±2%; E:T 20:1). This killing was confirmed to be HLA-restricted since the addition of an MHC class I blocking antibody diminished specific lysis (decrease from 44% to 26%). Finally, using individual peptides organized into minipools, we were able to identify 4 novel HLA class I-restricted epitope peptides.To assess the clinical relevance of CCNA1-directed T cells we assessed whether patients with AML had detectable circulating T cells and in 5 of 9 patients who had undergone an allogeneic hematopoietic stem cell transplant as disease treatment we could detect CCNA1-specific T cells (mean 117±52.8 SFC/2x105 expanded PBMCs), and detection of reactive cells correlated with a lower risk for relapse.In summary, we have identified CCNA1 as a suitable candidate antigen to target using immunotherapeutic approaches for future clinical trials of adoptively transferred T cells. DisclosuresLulla:Leukemia Texas: Membership on an entity's Board of Directors or advisory committees; ASH Scholar Award: Research Funding; Leukemia Texas Research grant: Research Funding; Junior Faculty sees funding-Baylor College of Medicine: Research Funding; Lymphoma SPORE: Research Funding; ASBMT Young Investigator Award: Research Funding. Tzannou:ViraCyte LLC: Consultancy. Heslop:Celgene: Research Funding; Cell Medica: Patents & Royalties: EBV specific T cells, Research Funding; Viracyte: Equity Ownership; Marker Therapeutics: Equity Ownership. Vera:Wilson Wolf Manufacturing: Consultancy; ViraCyte LLC: Equity Ownership; Marker Therapeutics: Equity Ownership. Leen:ViraCyte LLC: Equity Ownership.

Patients' NK cell stimulation with activated plasmacytoid dendritic cells increases dinutuximab-induced neuroblastoma killing.

Targeted immunotherapy has improved the outcome of patients with high-risk neuroblastoma (NB). However, immune escape of tumor cells still occurs and about 40% of NB patients relapse and die from their disease. We previously showed that natural killer (NK) cell stimulation by Toll-like receptor (TLR)-activated plasmacytoid dendritic cells (pDC) increases the efficacy of dinutuximab-based immunotherapy against NB cell lines via the TRAIL death-receptor pathway. With the aim to translate our findings into a novel adoptive therapy of TLR-activated pDC, we investigated the pDC/NK cell axis in NB patients undergoing dinutuximab-based immunotherapy. We show that pDC counts were low at the beginning of immunotherapy but reached normal levels over time. Blood NK cell counts were normal in all patients, although a high proportion of CD56bright CD16low/- cells was observed. The stimulation of patient's blood cells with a TLR9 ligand led to IFN-α production by pDC, and TRAIL expression on NK cell surface. Patient's NK cells expressed high levels of CD69 and TRAIL after stimulation with activated pDC. Both CD56bright CD16low/- and CD56dim CD16+ NK cells degranulated against autologous target cells in the presence of dinutuximab. Importantly, pDC-induced NK cell activation increased the dinutuximab mediated autologous killing of patient-derived NB cells. Altogether, our study demonstrates that TLR-activated pDC strongly increase the cytotoxic functions of NK cells in high-risk NB patients undergoing immunotherapy. These results, therefore, support pDC adoptive immunotherapy as a novel approach to decrease the risk of relapse in patients with high-risk NB.

Mass spectrometry-based identification of a B-cell maturation antigen-derived T-cell epitope for antigen-specific immunotherapy of multiple myeloma

The B-cell maturation antigen (BCMA) is currently being evaluated as promising tumor-associated surface antigen for T-cell-based immunotherapy approaches, such as CAR T cells and bispecific antibodies, in multiple myeloma (MM). Cytotoxic T cells bearing BCMA-specific T-cell receptors might further allow targeting HLA-presented antigens derived from the intracellular domain of BCMA. By analyzing a mass spectrometry-acquired immunopeptidome dataset of primary MM samples and MM cell lines for BCMA-derived HLA ligands, we identified the naturally presented HLA-B*18-restricted ligand P(BCMA)B*18. Additionally, P(BCMA)B*18 was identified on primary CLL samples, thereby expanding the range for possible applications. P(BCMA)B*18 induced multifunctional BCMA-specific cells de novo from naïve CD8+ T cells of healthy volunteers. These T cells exhibited antigen-specific lysis of autologous peptide-loaded cells. Even in the immunosuppressive context of MM, we detected spontaneous memory T-cell responses against P(BCMA)B*18 in patients. By applying CTLA-4 and PD-1 inhibition in vitro we induced multifunctional P(BCMA)B*18-specific CD8+ T cells in MM patients lacking preexisting BCMA-directed immune responses. Finally, we could show antigen-specific lysis of autologous peptide-loaded target cells and even MM.1S cells naturally presenting P(BCMA)B*18 using patient-derived P(BCMA)B*18-specific T cells. Hence, this BCMA-derived T-cell epitope represents a promising target for T-cell-based immunotherapy and monitoring following immunotherapy in B-cell malignancy patients.

Recurrent inflammatory disease caused by a heterozygous mutation in CD48

Recurrent inflammatory disease caused by a heterozygous mutation in CD48

A novel dendritic cell-based direct ex vivo assay for detection and enumeration of circulating antigen-specific human T cells.

Although a variety of assays have been used to examine T cell responses in vitro, standardized ex vivo detection of antigen-specific CD4+ T cells from human circulatory PBMCs remains constrained by low-dimensional characterization outputs and the need for polyclonal, mitogen-induced expansion methods to generate detectable response signals. To overcome these limitations, we developed a novel methodology utilizing antigen-pulsed autologous human dendritic target cells in a rapid and sensitive assay to accurately enumerate antigen-specific CD4+ T cell precursor frequency by multiparametric flow cytometry. With this approach, we demonstrate the ability to reproducibly quantitate poly-functional T cell responses following both primary and recall antigenic stimulation. Furthermore, this approach enables more comprehensive phenotypic profiling of circulating antigen-specific CD4+ T cells, providing valuable insights into the pre-existing polarization of antigen-specific T cells in humans. Combined, this approach permits sensitive and detailed ex vivo detection of antigen-specific CD4+ T cells delivering an important tool for advancing vaccine, immune-oncology and other therapeutic studies.

A single exercise bout augments adenovirus-specific T-cell mobilization and function

Adoptive transfer of virus-specific T-cells (VSTs) effectively treats viral infections following allogeneic hematopoietic stem cell transplantation (alloHSCT), but logistical difficulties have limited widespread availability of VSTs as a post-transplant therapeutic. A single exercise bout mobilizes VSTs specific for latent herpesviruses (i.e. CMV and EBV) to peripheral blood and augments their ex vivo expansion. We investigated whether exercise exerts similar effects on T-cells specific for a NON-latent virus such as adenovirus, which is a major contributor to infection-related morbidity and mortality after alloHSCT. Thirty minutes of cycling exercise increased circulating adenovirus-specific T-cells 2.0-fold and augmented their ex vivo expansion by ~33% compared to rest without altering antigen and MHC-specific autologous target cell killing capabilities. We conclude that exercise is a simple and economical adjuvant to boost the isolation and manufacture of therapeutic VSTs specific to latent and non-latent viruses from healthy donors.

The inhibitory checkpoint, PD-L2, is a target for effector T cells: Novel possibilities for immune therapy

ABSTRACTCell surface molecules of the B7/CD28 family play an important role in T-cell activation and tolerance. The relevance of the PD-1/PD-L1 pathway in cancer has been extensively studied whereas PD-L2 has received less attention. However, recently the expression of PD-L2 was described to be independently associated with clinical response in anti-PD1-treated cancer patients. Here, we investigated whether PD-L2 might represent a natural target that induces specific T cells. We identified spontaneous specific T-cell reactivity against two epitopes located in the signal peptide of PD-L2 from samples from patients with cancer as well as healthy individuals ex vivo. We characterized both CD8+ and CD4+ PD-L2-specific T cells. Interestingly, the epitope in PD-L2 that elicited the strongest response was equivalent to a potent HLA-A2-restricted epitope in PD-L1. Importantly, PD-L1-specific and PD-L2-specific T cells did not cross-react; therefore, they represent different T-cell antigens. Moreover, PD-L2-specific T cells reacted to autologous target cells depending on PD-L2 expression. These results suggested that activating PD-L2 specific T cells (e.g., by vaccination) might be an attractive strategy for anti-cancer immunotherapy. Accordingly, PD-L2 specific T cells can directly support anti-cancer immunity by killing of target cells, as well as, indirectly, by releasing pro-inflammatory cytokines at the microenvironment in response to PD-L2-expressing immune supressive cells.

A single exercise bout enhances the manufacture of viral-specific T-cells from healthy donors: implications for allogeneic adoptive transfer immunotherapy.

Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections remain a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). The adoptive transfer of donor-derived viral-specific cytotoxic T-cells (VSTs) is an effective treatment for controlling CMV and EBV infections after HSCT; however, new practical methods are required to augment the ex vivo manufacture of multi-VSTs from healthy donors. This study investigated the effects of a single exercise bout on the ex vivo manufacture of multi-VSTs. PBMCs isolated from healthy CMV/EBV seropositive participants before (PRE) and immediately after (POST) 30-minutes of cycling exercise were stimulated with CMV (pp65 and IE1) and EBV (LMP2A and BMLF1) peptides and expanded over 8 days. The number (fold difference from PRE) of T-cells specific for CMV pp65 (2.6), EBV LMP2A (2.5), and EBV BMLF1 (4.4) was greater among the VSTs expanded POST. VSTs expanded PRE and POST had similar phenotype characteristics and were equally capable of MHC-restricted killing of autologous target cells. We conclude that a single exercise bout enhances the manufacture of multi-VSTs from healthy donors without altering their phenotype or function and may serve as a simple and economical adjuvant to boost the production of multi-VSTs for allogeneic adoptive transfer immunotherapy.

A Single Bout Of Exercise Enhances The Ex Vivo Manufacture Of Viral-specific T-cells

Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections remain a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). The adoptive transfer of donor-derived viral-specific cytotoxic T-cells (CTLs) is an effective treatment for controlling CMV and EBV infections during HSCT. However, in many instances, the time taken to manufacture adequate numbers of CTLs from healthy donors is too long and requires the collection of large and impractical blood volumes. PURPOSE: To determine if a single exercise bout can augment the ex vivo manufacture of viral-specific T-cells from healthy donors. METHODS: Nine healthy CMV and EBV seropositive participants (mean ± SD age: 31.3 ± 3.3 years) completed a 30-min continuous cycling protocol at a workload corresponding to +15% of the individual blood lactate threshold. PBMCs (10x106) isolated before and immediately after exercise were stimulated with CMV (pp65 and IE1) and EBV (lmp2 and BMLF1) peptides and expanded over 8 days. At Day 8, viral-specific T-cells were enumerated using IFN-γ ELIPOST assays and the phenotypes of the expanded CTLs and viral-specific T-cells were determined by flow cytometry. RESULTS: Compared to CTLs expanded before exercise, the number of T-cells specific to CMV pp65, EBV lmp2, and EBV BMLF1 was markedly greater among the post-exercise expanded CTLs (fold-difference: CMVpp65: 2.6, EBV lmp-2: 2.5 and EBV BMLF-1: 4.4). Expanded CTLs predominantly consisted of effector memory (CD45RA-/CD62L-) and CD45RA+ effector memory (CD45RA+/CD62L-) T-cells, but no phenotypic differences were observed between CTLs expanded before and after exercise. Moreover, CTLs expanded before and after exercise were equally capable of killing viral-peptide pulsed autologous target cells in an MHC restricted manner. Viral-specific CTLs could not be expanded from a CMV/EBV seronegative participant, indicating that the augmenting effects of exercise are due to the priming and expansion of pre-existing memory T-cells. CONCLUSION: This is the first study to show that a single bout of exercise enhances the ex vivo manufacture of CMV and EBV-specific T-cells from healthy donors without altering their phenotype or function. Exercise may therefore serve as a safe adjuvant to improve viral-specific T-cell generation for HSCT.

Amplification Ex Vivo and Cytocidal Activity of Leukemia Tumor-Associated Antigen-Specific Cytotoxic T Lymphocytes

To explore the feasibility of amplifying the leukemia tumor associated antigens-specific cytotoxic T lymphocytes (TAA-CTL) ex vivo and to evaluate the cytotoxicity of TAA-CTL. The peripheral blood mononuclear cells were enriched by density gradient centrifugtion; TAA-CTL were generated by stimulation of PBMNC with peptide-pulsed DC at an effector-to-target ratio of 10:1; immunophenotype of TAA-CTL was analyzed by flow cytometry; cytotoxicity assay was used to evaluate the cytotoxic activity of TAA-CTL against peptide-pulsed autologous target cells (PHA-Blasts). TAA-CTL expanded from volunteer showed a mean expansion of 3.81±1.61, the phenotyping of the TAA-CTL was predominantly CD3+ (97.22±0.71)% with varying content of CD4+ (41.47±27.08)% and CD8+ (56.40±11.15)% T cells, it also contained few nature killer cells (0.50±0.31)% and rare residual B cells (0.14±0.20)%; the subpopulations of TAA-CTL and CTL were not statisticaly significantly different in the proportion (P>0.05); the detection of intracellular cytokines after stimulation with peptide showed that the secretion rates of IFN-γ and TNF-α in CD8+ TAA-CTL were (27.67±2.21)% and (34.2±0.71)%, while the secretion rates were (21.6±2.55)% and (9.97±3.44)% in CD4+ TAA-CTL. Compared with the CD8+ TAA-CTL group, the secretion rates of IFN-γ and TNF-α were (1.36±0.04)% and (5.58±0.03)% in CD8+ CTL, the rates of IFN-γ and TNF-α were (0.91±0.06)% and (1.60±0.07)% in CD4+ CTL. The secretions of IFN-γ and TNF-α in CTL were both significantly lower than those in TAA-CTL (P<0.01); the specific killing efficiency of the TAA-CTL against TAA-pulesd target cells were (77.00±1.00)%, (67.40±3.60)%, (60.55±2.45)% and (26.85±5.25)%, when the effecto-target ratios were 40:1, 20:1, 10:1 and 5:1, and there was negligible lysis of TAA-CTL for PHA-blast (P<0.01). TAA-CTL can be successfully induced and generated ex vivo from the healthy volunteer peripheral blood, and the TAA-CTL possess a specific killing activity.

Human CD56+ Cytotoxic Lung Lymphocytes Kill Autologous Lung Cells in Chronic Obstructive Pulmonary Disease

CD56+ natural killer (NK) and CD56+ T cells, from sputum or bronchoalveolar lavage of subjects with chronic obstructive pulmonary disease (COPD) are more cytotoxic to highly susceptible NK targets than those from control subjects. Whether the same is true in lung parenchyma, and if NK activity actually contributes to emphysema progression are unknown. To address these questions, we performed two types of experiments on lung tissue from clinically-indicated resections (n = 60). First, we used flow cytometry on fresh single-cell suspension to measure expression of cell-surface molecules (CD56, CD16, CD8, NKG2D and NKp44) on lung lymphocytes and of the 6D4 epitope common to MICA and MICB on lung epithelial (CD326+) cells. Second, we sequentially isolated CD56+, CD8+ and CD4+ lung lymphocytes, co-cultured each with autologous lung target cells, then determined apoptosis of individual target cells using Annexin-V and 7-AAD staining. Lung NK cells (CD56+ CD3−) and CD56+ T cells (CD56+ CD3+) were present in a range of frequencies that did not differ significantly between smokers without COPD and subjects with COPD. Lung NK cells had a predominantly “cytotoxic” CD56+ CD16+ phenotype; their co-expression of CD8 was common, but the percentage expressing CD8 fell as FEV1 % predicted decreased. Greater expression by autologous lung epithelial cells of the NKG2D ligands, MICA/MICB, but not expression by lung CD56+ cells of the activating receptor NKG2D, correlated inversely with FEV1 % predicted. Lung CD56+ lymphocytes, but not CD4+ or CD8+ conventional lung T cells, rapidly killed autologous lung cells without additional stimulation. Such natural cytotoxicity was increased in subjects with severe COPD and was unexplained in multiple regression analysis by age or cancer as indication for surgery. These data show that as spirometry worsens in COPD, CD56+ lung lymphocytes exhibit spontaneous cytotoxicity of autologous structural lung cells, supporting their potential role in emphysema progression.Trial RegistrationClinicalTrials.gov NCT00281229

T Cells Infiltrate the Liver and Kill Hepatocytes in HLA-B∗57:01-Associated Floxacillin-Induced Liver Injury

Drug-induced liver injury is a major safety issue. It can cause severe disease and is a common cause of the withdrawal of drugs from the pharmaceutical market. Recent studies have identified the HLA-B(∗)57:01 allele as a risk factor for floxacillin (FLUX)-induced liver injury and have suggested a role for cytotoxic CD8(+) T cells in the pathomechanism of liver injury caused by FLUX. This study aimed to confirm the importance of FLUX-reacting cytotoxic lymphocytes in the pathomechanism of liver injury and to dissect the involved mechanisms of cytotoxicity. IHC staining of a liver biopsy from a patient with FLUX-induced liver injury revealed periportal inflammation and the infiltration of cytotoxic CD3(+) CD8(+) lymphocytes into the liver. The infiltration of cytotoxic lymphocytes into the liver of a patient with FLUX-induced liver injury demonstrates the importance of FLUX-reacting T cells in the underlying pathomechanism. Cytotoxicity of FLUX-reacting T cells from 10 HLA-B(∗)57:01(+) healthy donors toward autologous target cells and HLA-B(∗)57:01-transduced hepatocytes was analyzed in vitro. Cytotoxicity of FLUX-reacting T cells was concentration dependent and required concentrations in the range of peak serum levels after FLUX administration. Killing of target cells was mediated by different cytotoxic mechanisms. Our findings emphasize the role of the adaptive immune system and especially of activated drug-reacting T cells in human leukocyte antigen-associated, drug-induced liver injury.

Combination of Epstein-Barr virus nuclear antigen 1, 3 and lytic antigen BZLF1 peptide pools allows fast and efficient stimulation of Epstein-Barr virus-specific T cells for adoptive immunotherapy

BackgroundEpstein-Barr virus (EBV) infection is a major cause of morbidity following hematopoietic stem cell transplantation. EBV-infected B cells may not respond to rituximab treatment and may lead to a life-threatening post-transplantation lymphoproliferative disorder. Adoptive cellular immunotherapy using EBV-lymphoblastoid cell lines (LCL) as stimulating antigen has proved effective in restoring specific immunity. However, EBV presents several immunodominant antigens, and developing a swift and effective clinical-grade immunotherapy relies on the definition of a Good Manufacturing Practices (GMP) universal stimulating antigen. MethodsPeripheral blood mononuclear cells (PBMCs) from six donors with a cellular immune response against EBV were immunoselected after stimulation with a new EBV antigen associated with an EBNA3 peptide pool. ResultsAfter immunoselection, a mean of 0.53 ± 0.25 × 106 cells was recovered consisting of a mean of 24.77 ± 18.01% CD4+-secreting interferon (IFN)-γ and 51.42 ± 26.92% CD8+-secreting IFN-γ. The T memory stem cell sub-population was identified. EBV-specific T cells were expanded in vitro, and their ability to secrete IFN-γ and to proliferate after re-stimulation with EBV antigen was confirmed. A specific lysis was observed against autologous target cells pulsed with EBV peptide pools (57.6 ± 11.5%) and against autologous EBV-LCL (18.3 ± 7.3%). A mean decrease of 94.7 ± 3.3% in alloreactivity against third-party donor mononuclear cells with EBV-specific T cells was observed compared with PBMCs before selection. ConclusionsOur results show that a combination of peptide pools including EBNA3 is needed to generate EBV-specific T cells with good specific cytotoxicity and devoid of alloreactivity, but as yet GMP grade is not fully achieved.

Targeting concatenated HIV antigens to human CD40 expands a broad repertoire of multifunctional CD4+ and CD8+ T cells

Targeting HIV antigens directly to dendritic cells using monoclonal antibodies against cell-surface receptors has been shown to evoke potent cellular immunity in animal models. The objective of this study was to configure an anti-human CD40 antibody fused to a string of five highly conserved CD4 and CD8 T-cell epitope-rich regions of HIV-1 Gag, Nef and Pol (αCD40.HIV5pep), and then to demonstrate the capacity of this candidate therapeutic vaccine to target these HIV peptide antigens to human dendritic cells to expand functional HIV-specific T cells. Antigen-specific cytokine production using intracellular flow cytometry and multiplex bead-based assay, and suppression of viral inhibition, were used to characterize the T cells expanded by αCD40.HIV5pep from HIV-infected patient peripheral blood mononuclear cell (PBMC) and dendritic cell/T-cell co-cultures. This candidate vaccine expands memory CD4 and CD8 T cells specific to multiple epitopes within all five peptide regions across a wide range of major histocompatibility complex (MHC) haplotypes from HIV-infected patient PBMC and dendritic cell/T-cell co-cultures. These in vitro expanded HIV antigen-specific CD4 and CD8 T cells produce multiple cytokines and chemokines. αCD40.HIV5pep-expanded CD8 T cells have characteristics of cytotoxic effector cells and are able to kill autologous target cells and suppress HIV-1 replication in vitro. Our data demonstrate the therapeutic potential of this CD40-targeting HIV candidate vaccine in inducing a broad repertoire of multifunctional T cells in patients.

Immune response and survival of refractory cancer patients who received TGF-β2 antisense/GM-CSF gene modified autologous tumor cell (TAG) vaccine

TAG vaccine is a novel 'triad vaccine' that involves transfection of autologous tumor with a dual plasmid, TGFβ2 antisense gene and GM-CSF gene. Patients with advanced cancer who failed standard therapy were treated. IFN-γ ELISPOT analysis (Enzyme-Linked Immunospot Assay for Interferon Gamma) using TAG autologous vaccine target cells was performed prior to vaccination and at week 12 after the third vaccination. The purpose of this assessment was to correlate the IFN-γ ELISPOT immune response with long-term survival of advanced cancer patients who received TAG vaccination. Twenty-three of 28 patients received ≥ 3 TAG vaccinations (two patients withdrew consent and three had disease progression prior to the third vaccination). Eleven patients demonstrated a positive ELISPOT response (>10 spots and ≥ 2 × baseline) at week 12 and 12 patients did not (P=0.002). Median survival from time of treatment between ELISPOT-positive and -negative groups was significantly different (550 vs 159 days, P=0.036), as was median survival from the time of procurement (627 vs 257 days, respectively, P=0.043). In conclusion, the IFN-γ ELISPOT assay may provide an effective measure of immune response following treatment with 'triad vaccines', but additional patient numbers and/or other immune modulatory parameters are necessary for future testing.

Stimulation of Human EBV- and CMV-Specific Cytolytic Effector Function Using Allogeneic HLA Molecules

Viral infection is a major cause of morbidity and mortality, and there are few therapeutic options available to augment a virus-specific T cell response. Although allo-HLA cross-reactivity from virus-specific memory T cells is common, it is unclear whether priming with specific allogeneic cells could conversely elicit a viral peptide/self-HLA restricted cytotoxic T cell response in humans. First, we used the previously described allo-HLA-B*44:02 cross-reactivity of EBV peptide/HLA-B8 restricted T cells, to determine whether allogeneic HLA stimulation can elicit a cytolytic immune response against EBV. HLA-B8(+) HLA-B44(-) EBV-seropositive PBMCs were stimulated with either HLA-B*44:02(+) or HLA-B*44:03(+) mismatched irradiated PBMCs in a 7-10 d MLR. The allo-HLA stimulated responder cells were then evaluated for cytotoxicity using EBV peptide loaded autologous target cells and unloaded HLA-B8(+) EBV LCL target cells. PBMCs from EBV-seropositive donors gained EBV-specific cytolytic effector function following specific allo-HLA stimulation. Finally, we also elicited cytolytic CMV-specific responses using specific allogeneic cell stimulation, to confirm that this technique can be used to elicit viral peptide/self-HLA restricted responses even from nonpublic TCR responses. Allogeneic cell stimulation used as a cell therapy may be a potential tool to augment an antiviral T cell response in patients with EBV or CMV infection.