Combined treatment with allogeneic Epstein-Barr- and human polyomavirus 1 specific T-cells in progressive multifocal leukoencephalopathy and EBV infection: a case report.

Opportunistic viral infections in individuals with severe immunodeficiency can lead to fatal conditions such as progressive multifocal leukoencephalopathy (PML), for which treatment options are limited. These infections pose significant risks, especially when co-infections with other viruses occur. We describe a combined therapy approach using directly isolated allogeneic Human Polyomavirus 1 (also known as BKV) and Epstein-Barr virus (EBV) specific cytotoxic T-cells for the treatment of PML in conjunction with identified EBV in the cerebrospinal fluid (CSF) of a male patient infected with human immunodeficiency virus (HIV). A 53-year-old HIV-positive male, recently diagnosed with PML, presented with rapidly worsening symptoms, including ataxia, tetraparesis, dysarthria, and dysphagia, leading to respiratory failure. The patient developed PML even after commencing highly active antiretroviral therapy (HAART) 3 months prior. Brain magnetic resonance imaging (MRI) revealed multifocal demyelination lesions involving the posterior fossa and right thalamus suggestive of PML. In addition to the detection of human polyomavirus 2 (also known as JCV), analysis of CSF showed positive results for EBV deoxyribonucleic acid (DNA). His neurological condition markedly deteriorated over the following 2 months. Based on MRI, there was no evidence of Immune Reconstitution Inflammatory Syndrome contributing to this decline. The patient did not have endogenous virus-specific T-cells. We initiated an allogeneic, partially human leukocyte antigen-matched transfer of EBV and utilizing the cross-reactivity between BKV and JCV-BKV specific T-cells. This intervention led to notable neurological improvement and partial resolution of the MRI lesions within 6 weeks. Our case of a patient with acquired immune deficiency syndrome demonstrates that PML and concurrent EBV co-infection can still occur despite undergoing HAART treatment. This innovative experimental therapy, involving a combination of virus-specific T-cells, was demonstrated to be an effective treatment option in this patient.

In Vitro Profiling of Commonly Used Post-transplant Immunosuppressants Reveals Distinct Impact on Antiviral T-cell Immunity Towards CMV.

Infectious complications, including widespread human cytomegalovirus (CMV) disease, frequently occur after hematopoietic stem cell and solid organ transplantation due to immunosuppressive treatment causing impairment of T-cell immunity. Therefore, in-depth analysis of the impact of immunosuppressants on antiviral T cells is needed. We analyzed the impact of mTOR inhibitors sirolimus (SIR/S) and everolimus (EVR/E), calcineurin inhibitor tacrolimus (TAC/T), purine synthesis inhibitor mycophenolic acid (MPA/M), glucocorticoid prednisolone (PRE/P) and common double (T+S/E/M/P) and triple (T+S/E/M+P) combinations on antiviral T-cell functionality. T-cell activation and effector molecule production upon antigenic stimulation was impaired in presence of T+P and triple combinations. SIR, EVR and MPA exclusively inhibited T-cell proliferation, TAC inhibited activation and cytokine production and PRE inhibited various aspects of T-cell functionality including cytotoxicity. This was reflected in an in vitro infection model, where elimination of CMV-infected human fibroblasts by CMV-specific T cells was reduced in presence of PRE and all triple combinations. CMV-specific memory T cells were inhibited by TAC and PRE, which was also reflected with double (T+P) and triple combinations. EBV- and SARS-CoV-2-specific T cells were similarly affected. These results highlight the need to optimize immune monitoring to identify patients who may benefit from individually tailored immunosuppression.

Human cytomegalovirus exploits STING signaling and counteracts IFN/ISG induction to facilitate infection of dendritic cells.

Human cytomegalovirus (HCMV) is a widespread pathogen that in immunocompromised hosts can cause life-threatening disease. Studying HCMV-exposed monocyte-derived dendritic cells by single-cell RNA sequencing, we observe that most cells are entered by the virus, whereas less than 30% of them initiate viral gene expression. Increased viral gene expression is associated with activation of the stimulator of interferon genes (STING) that usually induces anti-viral interferon responses, and with the induction of several pro- (RHOB, HSP1A1, DNAJB1) and anti-viral (RNF213, TNFSF10, IFI16) genes. Upon progression of infection, interferon-beta but not interferon-lambda transcription is inhibited. Similarly, interferon-stimulated gene expression is initially induced and then shut off, thus further promoting productive infection. Monocyte-derived dendritic cells are composed of 3 subsets, with one being especially susceptible to HCMV. In conclusion, HCMV permissiveness of monocyte-derived dendritic cells depends on complex interactions between virus sensing, regulation of the interferon response, and viral gene expression.

Identification of novel canonical and cryptic HCMV-specific T-cell epitopes for HLA-A∗03 and HLA-B∗15 via peptide-PRISM.

ABSTRACT: Human cytomegalovirus (HCMV) reactivation poses a substantial risk to patients receiving tranplants. Effective risk stratification and vaccine development is hampered by a lack of HCMV-derived immunogenic peptides in patients with common HLA-A∗03:01 and HLA-B∗15:01 haplotypes. This study aimed to discover novel HCMV immunogenic peptides for these haplotypes by combining ribosome sequencing (Ribo-seq) and mass spectrometry with state-of-the-art computational tools, Peptide-PRISM and Probabilistic Inference of Codon Activities by an EM Algorithm. Furthermore, using machine learning, an algorithm was developed to predict immunogenicity based on translational activity, binding affinity, and peptide localization within small open reading frames to identify the most promising peptides for in vitro validation. Immunogenicity of these peptides was subsequently tested by analyzing peptide-specific T-cell responses of HCMV-seropositive and -seronegative healthy donors as well as patients with transplants. This resulted in the direct identification of 3 canonical and 1 cryptic HLA-A∗03-restricted immunogenic peptides as well as 5 canonical and 1 cryptic HLA-B∗15-restricted immunogenic peptide, with a specific interferon gamma-positive (IFN-γ+)/CD8+ T-cell response of ≥0.02%. High T-cell responses were detected against 2 HLA-A∗03-restricted and 3 HLA-B∗15-restricted canonical peptides with frequencies of up to 8.77% IFN-γ+/CD8+ T cells in patients after allogeneic stem cell transplantation. Therefore, our comprehensive strategy establishes a framework for efficient identification of novel immunogenic peptides from both existing and novel Ribo-seq data sets.

CAR-Ts redirected against the Thomsen-Friedenreich antigen CD176 mediate specific elimination of malignant cells from leukemia and solid tumors.

Introduction: Chimeric antigen receptor-engineered T cells (CAR-Ts) are investigated in various clinical trials for the treatment of cancer entities beyond hematologic malignancies. A major hurdle is the identification of a target antigen with high expression on the tumor but no expression on healthy cells, since "on-target/off-tumor" cytotoxicity is usually intolerable. Approximately 90% of carcinomas and leukemias are positive for the Thomsen-Friedenreich carbohydrate antigen CD176, which is associated with tumor progression, metastasis and therapy resistance. In contrast, CD176 is not accessible for ligand binding on healthy cells due to prolongation by carbohydrate chains or sialylation. Thus, no "on-target/off-tumor" cytotoxicity and low probability of antigen escape is expected for corresponding CD176-CAR-Ts. Methods: Using the anti-CD176 monoclonal antibody (mAb) Nemod-TF2, the presence of CD176 was evaluated on multiple healthy or cancerous tissues and cells. To target CD176, we generated two different 2nd generation CD176-CAR constructs differing in spacer length. Their specificity for CD176 was tested in reporter cells as well as primary CD8+ T cells upon co-cultivation with CD176+ tumor cell lines as models for CD176+ blood and solid cancer entities, as well as after unmasking CD176 on healthy cells by vibrio cholerae neuraminidase (VCN) treatment. Following that, both CD176-CARs were thoroughly examined for their ability to initiate target-specific T-cell signaling and activation, cytokine release, as well as cytotoxicity. Results: Specific expression of CD176 was detected on primary tumor tissues as well as on cell lines from corresponding blood and solid cancer entities. CD176-CARs mediated T-cell signaling (NF-κB activation) and T-cell activation (CD69, CD137 expression) upon recognition of CD176+ cancer cell lines and unmasked CD176, whereby a short spacer enabled superior target recognition. Importantly, they also released effector molecules (e.g. interferon-γ, granzyme B and perforin), mediated cytotoxicity against CD176+ cancer cells, and maintained functionality upon repetitive antigen stimulation. Here, CD176L-CAR-Ts exhibited slightly higher proliferation and mediator-release capacities. Since both CD176-CAR-Ts did not react towards CD176- control cells, their response proved to be target-specific. Discussion: Genetically engineered CD176-CAR-Ts specifically recognize CD176 which is widely expressed on cancer cells. Since CD176 is masked on most healthy cells, this antigen and the corresponding CAR-Ts represent a promising approach for the treatment of various blood and solid cancers while avoiding "on-target/off-tumor" cytotoxicity.

Adoptive Allogeneic T-Cell Therapy Improves the Clinical Outcome of JC Virus Granule Cell Neuronopathy: A Case Report.

OBJECTIVES: JC virus granule cell neuronopathy is a potentially fatal otherwise highly disabling disease without an approved therapeutic option. This case report presents the positive record to T-cell therapy in JC virus granule cell neuronopathy. METHODS: The patient represented with subacute cerebellar symptoms. Diagnosis of JC virus granule cell neuronopathy was made because of infratentorially accentuated brain volume atrophy shown by brain MRI and the detection of JC virus DNA in the CSF. RESULTS: Six doses of virus-specific T cells were administered. Within 12 months after therapy initiation, the patient showed clear clinical benefit with improvement of symptoms, and JC viral DNA load significantly declined. DISCUSSION: We present the case report of a positive response to T-cell therapy in JC virus granule cell neuronopathy, leading to an improvement of symptoms.

Patient-tailored adoptive immunotherapy with EBV-specific T cells from related and unrelated donors.

BACKGROUNDAdoptive transfer of EBV-specific T cells can restore specific immunity in immunocompromised patients with EBV-associated complications.METHODSWe provide results of a personalized T cell manufacturing program evaluating donor, patient, T cell product, and outcome data. Patient-tailored clinical-grade EBV-specific cytotoxic T lymphocyte (EBV-CTL) products from stem cell donors (SCDs), related third-party donors (TPDs), or unrelated TPDs from the allogeneic T cell donor registry (alloCELL) at Hannover Medical School were manufactured by immunomagnetic selection using a CliniMACS Plus or Prodigy device and the EBV PepTivators EBNA-1 and Select. Consecutive manufacturing processes were evaluated, and patient outcome and side effects were retrieved by retrospective chart analysis.RESULTSForty clinical-grade EBV-CTL products from SCDs, related TPDs, or unrelated TPDs were generated for 37 patients with refractory EBV infections or EBV-associated malignancies with and without a history of transplantation, within 5 days (median) after donor identification. Thirty-four patients received 1-14 EBV-CTL products (fresh and cryopreserved). EBV-CTL transfer led to a complete response in 20 of 29 patients who were evaluated for clinical response. No infusion-related toxicity was reported. EBV-specific T cells in patients' blood were detectable in 16 of 18 monitored patients (89%) after transfer, and their presence correlated with clinical response.CONCLUSIONPersonalized clinical-grade manufacture of EBV-CTL products via immunomagnetic selection from SCDs, related TPDs, or unrelated TPDs in a timely manner is feasible. Overall, EBV-CTLs were clinically effective and well tolerated. Our data suggest EBV-CTL transfer as a promising therapeutic approach for immunocompromised patients with refractory EBV-associated diseases beyond HSCT, as well as patients with preexisting organ dysfunction.TRIAL REGISTRATIONNot applicable.FUNDINGThis study was funded in part by the German Research Foundation (DFG, 158989968/SFB 900), the Deutsche Kinderkrebsstiftung (DKS 2013.09), Wilhelm-Sander-Stiftung (reference 2015.097.1), Ellen-Schmidt-Program of Hannover Medical School, and German Federal Ministry of Education and Research (reference 01EO0802).

Long-Term Follow-Up after Adoptive Transfer of BK-Virus-Specific T Cells in Hematopoietic Stem Cell Transplant Recipients.

The BK virus (BKV) causes severe hemorrhagic cystitis in hematopoietic stem cell transplant (HSCT) recipients. To eliminate reactivated BKV, symptomatic patients can be treated with a reduction of the immunosuppressive therapy, with the antiviral drug cidofovir, or with virus-specific T cells (VSTs). In the current study, we compared the effect of VSTs to other treatment options, following up specific T cells using interferon-gamma ELISpot assay. We observed BKV large T-specific cellular responses in 12 out of 17 HSCT recipients with BKV-related cystitis (71%). In recipients treated with VSTs, 6 out of 7 showed specific T-cell responses, and that number in those without VSTs was 6 out of 10. In comparison, 27 out of 50 healthy controls (54%) responded. In HSCT recipients treated for BKV-related cystitis, absolute CD4+ T-cell numbers and renal function correlated with BKV-specific cellular responses (p = 0.03 and 0.01, respectively). In one patient, BKV-specific cellular immunity could already be detected at baseline, on day 35 after HSCT and prior to VSTs, and remained increased until day 226 after VSTs (78 vs. 7 spots increment). In conclusion, the ELISpot appears to be suitable to sensitively monitor BKV-specific cellular immunity in HSCT recipients, even early after transplantation or in the long term after VSTs.

Rapid and sustained T cell-based immunotherapy against invasive fungal disease via a combined two step procedure.

Introduction: Aspergillus fumigatus (Asp) infections constitute a major cause of morbidity and mortality in patients following allogeneic hematopoietic stem cell transplantation (HSCT). In the context of insufficient host immunity, antifungal drugs show only limited efficacy. Faster and increased T-cell reconstitution correlated with a favorable outcome and a cell-based therapy approach strongly indicated successful clearance of fungal infections. Nevertheless, complex and cost- or time-intensive protocols hampered their implementation into clinical application. Methods: To facilitate the clinical-scale manufacturing process of Aspergillus fumigatus-specific T cells (ATCs) and to enable immediate (within 24 hours) and sustained (12 days later) treatment of patients with invasive aspergillosis (IA), we adapted and combined two complementary good manufacturing practice (GMP)-compliant approaches, i) the direct magnetic enrichment of Interferon-gamma (IFN-γ) secreting ATCs using the small-scale Cytokine Secretion Assay (CSA) and ii) a short-term in vitro T-cell culture expansion (STE), respectively. We further compared stimulation with two standardized and commercially available products: Asp-lysate and a pool of overlapping peptides derived from different Asp-proteins (PepMix). Results: For the fast CSA-based approach we detected IFN-γ+ ATCs after Asp-lysate- as well as PepMix-stimulation but with a significantly higher enrichment efficiency for stimulation with the Asp-lysate when compared to the PepMix. In contrast, the STE approach resulted in comparably high ATC expansion rates by using Asp-lysate or PepMix. Independent of the stimulus, predominantly CD4+ helper T cells with a central-memory phenotype were expanded while CD8+ T cells mainly showed an effector-memory phenotype. ATCs were highly functional and cytotoxic as determined by secretion of granzyme-B and IFN-γ. Discussion: For patients with IA, the immediate adoptive transfer of IFN-γ+ ATCs followed by the administration of short-term in vitro expanded ATCs from the same donor, might be a promising therapeutic option to improve the clinical outcome.

Reinforcement of cell-mediated immunity driven by tumor-associated Epstein-Barr virus (EBV)-specific T cells during targeted B-cell therapy with rituximab.

Introduction: In immunocompromised patients, Epstein-Barr virus (EBV) infection or reactivation is associated with increased morbidity and mortality, including the development of B-cell lymphomas. The first-line treatment consists of reduction of immunosuppression and administration of rituximab (anti-CD20 antibody). Furthermore, the presence of EBV-specific T cells against latent EBV proteins is crucial for the control of EBV-associated diseases. Therefore, in addition to effective treatment strategies, appropriate monitoring of T cells of high-risk patients is of great importance for improving clinical outcome. In this study, we hypothesized that rituximab-mediated lysis of malignant EBV-infected B cells leads to the release and presentation of EBV-associated antigens and results in an augmentation of EBV-specific effector memory T-cell responses. Methods: EBV-infected B lymphoblastoid cell lines (B-LCLs) were used as a model for EBV-associated lymphomas, which are capable of expressing latency stage II and III EBV proteins present in all known EBV-positive malignant cells. Rituximab was administered to obtain cell lysates containing EBV antigens (ACEBV). Efficiency of cross-presentation of EBV-antigen by B-LCLs compared to cross-presentation by professional antigen presenting cells (APCs) such as dendritic cells (DCs) and B cells was investigated by in vitro T-cell immunoassays. Deep T-cell profiling of the tumor-reactive EBV-specific T cells in terms of activation, exhaustion, target cell killing, and cytokine profile was performed, assessing the expression of T-cell differentiation and activation markers as well as regulatory and cytotoxic molecules by interferon-γ (IFN-γ) EliSpot assay, multicolor flow cytometry, and multiplex analyses. Results: By inhibiting parts of the cross-presentation pathway, B-LCLs were shown to cross-present obtained exogenous ACEBV-derived antigens mainly through major histocompatibility complex (MHC) class I molecules. This mechanism is comparable to that for DCs and B cells and resulted in a strong EBV-specific CD8+ cytotoxic T-cell response. Stimulation with ACEBV-loaded APCs also led to the activation of CD4+ T helper cells, suggesting that longer peptide fragments are processed via the classical MHC class II pathway. In addition, B-LCLs were also found to be able to take up exogenous antigens from surrounding cells by endocytosis leading to induction of EBV-specific T-cell responses although to a much lesser extent than cross-presentation of ACEBV-derived antigens. Increased expression of activation markers CD25, CD71 and CD137 were detected on EBV-specific T cells stimulated with ACEBV-loaded APCs, which showed high proliferative and cytotoxic capacity as indicated by enhanced EBV-specific frequencies and increased secretion levels of cytotoxic effector molecules (e.g. IFN-γ, granzyme B, perforin, and granulysin). Expression of the regulatory proteins PD-1 and Tim-3 was induced but had no negative impact on effector T-cell functions. Conclusion: In this study, we showed for the first time that rituximab-mediated lysis of EBV-infected tumor cells can efficiently boost EBV-specific endogenous effector memory T-cell responses through cross-presentation of EBV-derived antigens. This promotes the restoration of antiviral cellular immunity and presents an efficient mechanism to improve the treatment of CD20+ EBV-associated malignancies. This effect is also conceivable for other therapeutic antibodies or even for therapeutically applied unmodified or genetically modified T cells, which lead to the release of tumor antigens after specific cell lysis.

Adenoviral Penton and Hexon Proteins Are Equivalent Immunogenic Targets of Virus-Specific T Cells after Hematopoietic Stem Cell Transplantation in Children.

Human adenovirus (HAdV) infection is a serious complication that can lead to significant morbidity and mortality, especially in immunocompromised pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT). Control and elimination of HAdV requires the presence of the respective antiviral T cells, and adoptive transfer of virus-specific T cells has become an important new treatment option for patients refractory to antiviral treatment. Although the adenoviral capsid protein hexon is known to be a major immunodominant T cell target across HAdV species, up to 30% of HAdV-seropositive donors show no T cell responses to the overlapping peptide pool spanning the entire protein. Our group recently verified the capsid protein penton as a second immunodominant target in HAdV infection. Here we aimed to investigate the prevalence of both penton-specific and hexon-specific HAdV T cells and their impact in virus control after HSCT. We analyzed the prevalence and characteristics of HAdV-specific T cells in 33 consecutive pediatric patients with HAdV reactivation following allogeneic HSCT and correlated them with viral load analysis. Our study demonstrates that penton is an important immunodominant target antigen of HAdV reactivation/ infection after HSCT in most patients. We demonstrate that in the majority of patients, both penton- and hexon-specific T cells appear at similar time intervals after transplantation. Despite the prevalence for either hexon-specific or penton-specific T cells in individual patients, we were unable to attribute the predominance to specific HLA types or HAdV serotypes. The occurrence of HAdV-specific T cells was closely linked to viral control, arguing for immune monitoring strategies to tailor antiviral treatment and adoptive T cell therapy.

Chronic Active Epstein-Barr Virus (EBV) Infection Controlled by Allogeneic Stem Cell Transplantation and EBV-Specific T Cells.

We report sustained remission of chronic active Epstein-Barr virus (EBV) infection in a 27-year-old female patient treated with third-party EBV-specific T cells followed by allogeneic hematopoietic stem cell transplantation (HSCT). The viremia cleared after administration of anti-T-lymphocyte globulin for graft-versus-host disease (GvHD) prophylaxis. Subsequent expansion of EBV-infected host T cells was controlled by transfusion of donor-derived EBV-specific T cells.

αß and γδ T-cell responses to Epstein-Barr Virus: insights in immunocompetence, immune failure and therapeutic augmentation in transplant patients.

Epstein-Barr Virus (EBV) is a human gamma herpes virus, which causes several diseases in immunocompetent (mononucleosis, chronic fatigue syndrome, gastric cancer, endemic Burkitt's lymphoma, head and neck cancer) and immunosuppressed (post-transplant lymphoproliferative disease, EBV-associated soft tissue tumors) patients. It elicits a complex humoral and cellular immune response with both innate and adaptive immune components. Substantial progress has been made in understanding the interplay of immune cells in EBV-associated diseases in recent years, and several therapeutic approaches have been developed to augment cellular immunity toward EBV for control of EBV-associated malignancy. This review will focus on recent developments in immunosuppressed transplant recipients.

Targeted proteomics identifies circulating biomarkers associated with active COVID-19 and post-COVID-19.

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic is caused by the highly infectious Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). There is an urgent need for biomarkers that will help in better stratification of patients and contribute to personalized treatments. We performed targeted proteomics using the Olink platform and systematically investigated protein concentrations in 350 hospitalized COVID-19 patients, 186 post-COVID-19 individuals, and 61 healthy individuals from 3 independent cohorts. Results revealed a signature of acute SARS-CoV-2 infection, which is represented by inflammatory biomarkers, chemokines and complement-related factors. Furthermore, the circulating proteome is still significantly affected in post-COVID-19 samples several weeks after infection. Post-COVID-19 individuals are characterized by upregulation of mediators of the tumor necrosis (TNF)-α signaling pathways and proteins related to transforming growth factor (TGF)-ß. In addition, the circulating proteome is able to differentiate between patients with different COVID-19 disease severities, and is associated with the time after infection. These results provide important insights into changes induced by SARS-CoV-2 infection at the proteomic level by integrating several cohorts to obtain a large disease spectrum, including variation in disease severity and time after infection. These findings could guide the development of host-directed therapy in COVID-19.

Stem Cell Therapy in Neuroimmunological Diseases and Its Potential Neuroimmunological Complications.

Background: Since the 1990s, transplantations of hematopoietic and mesenchymal stem cells (HSCT and MSCT) and dendritic cell (DCT) have been investigated for the treatment of neurological autoimmune disorders (NADs). With the growing number of transplanted patients, awareness of neuroimmunolgical complications has increased. Therefore, an overview of SCT for the most common NADs and reports of secondary immunity after SCT is provided. Methods: For this narrative review, a literature search of the PubMed database was performed. A total of 86 articles reporting on different SCTs in NADs and 61 articles dealing with immune-mediated neurological complications after SCT were included. For multiple sclerosis (MS), only registered trials and phase I/II or II studies were considered, whereas all available articles on other disorders were included. The different transplantation procedures and efficacy and safety data are presented. Results: In MS patients, beneficial effects of HSCT, MSCT, and DCT with a decrease in disability and stabilization of disease activity have been reported. These effects were also shown in other NADs mainly in case reports. In seven of 132 reported patients with immune-mediated neurological complications, the outcome was fatal. Conclusions: Phase III trials are ongoing for MS, but the role of SCT in other NADs is currently limited to refractory patients due to occasional serious complications.

GMP-Compliant Manufacturing of TRUCKs: CAR T Cells targeting GD2 and Releasing Inducible IL-18.

Chimeric antigen receptor (CAR)-engineered T cells can be highly effective in the treatment of hematological malignancies, but mostly fail in the treatment of solid tumors. Thus, approaches using 4th advanced CAR T cells secreting immunomodulatory cytokines upon CAR signaling, known as TRUCKs ("T cells redirected for universal cytokine-mediated killing"), are currently under investigation. Based on our previous development and validation of automated and closed processing for GMP-compliant manufacturing of CAR T cells, we here present the proof of feasibility for translation of this method to TRUCKs. We generated IL-18-secreting TRUCKs targeting the tumor antigen GD2 using the CliniMACS Prodigy® system using a recently described "all-in-one" lentiviral vector combining constitutive anti-GD2 CAR expression and inducible IL-18. Starting with 0.84 x 108 and 0.91 x 108 T cells after enrichment of CD4+ and CD8+ we reached 68.3-fold and 71.4-fold T cell expansion rates, respectively, in two independent runs. Transduction efficiencies of 77.7% and 55.1% was obtained, and yields of 4.5 x 109 and 3.6 x 109 engineered T cells from the two donors, respectively, within 12 days. Preclinical characterization demonstrated antigen-specific GD2-CAR mediated activation after co-cultivation with GD2-expressing target cells. The functional capacities of the clinical-scale manufactured TRUCKs were similar to TRUCKs generated in laboratory-scale and were not impeded by cryopreservation. IL-18 TRUCKs were activated in an antigen-specific manner by co-cultivation with GD2-expressing target cells indicated by an increased expression of activation markers (e.g. CD25, CD69) on both CD4+ and CD8+ T cells and an enhanced release of pro-inflammatory cytokines and cytolytic mediators (e.g. IL-2, granzyme B, IFN-γ, perforin, TNF-α). Manufactured TRUCKs showed a specific cytotoxicity towards GD2-expressing target cells indicated by lactate dehydrogenase (LDH) release, a decrease of target cell numbers, microscopic detection of cytotoxic clusters and detachment of target cells in real-time impedance measurements (xCELLigence). Following antigen-specific CAR activation of TRUCKs, CAR-triggered release IL-18 was induced, and the cytokine was biologically active, as demonstrated in migration assays revealing specific attraction of monocytes and NK cells by supernatants of TRUCKs co-cultured with GD2-expressing target cells. In conclusion, GMP-compliant manufacturing of TRUCKs is feasible and delivers high quality T cell products.

Enhancement of Antiviral T-Cell Responses by Vitamin C Suggests New Strategies to Improve Manufacturing of Virus-Specific T Cells for Adoptive Immunotherapy.

Allogeneic and autologous transplantation of hematopoietic stem cells (HSCT) are being routinely used to treat patients with leukemia and lymphoma. Due to the required immunosuppression after stem cell transplantation, infection and reactivation by viruses are life-threatening complications. In recent years, adoptive transfer using virus-specific T cells (VSTs) has emerged as alternative to conventional therapies. Since vitamins are described to influence the immune system and its cellular components, the aim of this study was to examine whether vitamins modulate VST function and thereby enable an improvement of therapy. For that, we investigated the impact of vitamin C and D on the functionality of cytomegalovirus (CMV)-specific T cells isolated from CMV-seropositive healthy donors. We were able to show that vitamin C increases the expansion and activation state of CMV-specific T cells, and an increased influence of vitamin C was observed on cells isolated from male donors and donors above 40 years of age. A higher frequency of the terminally differentiated effector memory CD8+ T-cell population in these donors indicates a connection between these cells and the enhanced response to vitamin C. Thus, here we provide insights into the impact of vitamin C on cytotoxic T cells as well as possible additional selection criteria and strategies to improve VST functionality.

Rapid Manufacturing of Highly Cytotoxic Clinical-Grade SARS-CoV-2-specific T Cell Products Covering SARS-CoV-2 and Its Variants for Adoptive T Cell Therapy.

Objectives: Evaluation of the feasibility of SARS-CoV-2-specific T cell manufacturing for adoptive T cell transfer in COVID-19 patients at risk to develop severe disease. Methods: Antiviral SARS-CoV-2-specific T cells were detected in blood of convalescent COVID-19 patients following stimulation with PepTivator SARS-CoV-2 Select using Interferon-gamma Enzyme-Linked Immunospot (IFN-γ ELISpot), SARS-CoV-2 T Cell Analysis Kit (Whole Blood) and Cytokine Secretion Assay (CSA) and were characterized with respect to memory phenotype, activation state and cytotoxic potential by multicolor flow cytometry, quantitative real-time PCR and multiplex analyses. Clinical-grade SARS-CoV-2-specific T cell products were generated by stimulation with MACS GMP PepTivator SARS-CoV-2 Select using CliniMACS Prodigy and CliniMACS Cytokine Capture System (IFN-gamma) (CCS). Functionality of enriched T cells was investigated in cytotoxicity assays and by multiplex analysis of secreted cytotoxic molecules upon target recognition. Results: Donor screening via IFN-γ ELISpot allows for pre-selection of potential donors for generation of SARS-CoV-2-specific T cells. Antiviral T cells reactive against PepTivator SARS-CoV-2 Select could be magnetically enriched from peripheral blood of convalescent COVID-19 patients by small-scale CSA resembling the clinical-grade CCS manufacturing process and showed an activated and cytotoxic T cell phenotype. Four clinical-grade SARS-CoV-2-specific T cell products were successfully generated with sufficient cell numbers and purities comparable to those observed in donor pretesting via CSA. The T cells in the generated products were shown to be capable to replicate, specifically recognize and kill target cells in vitro and secrete cytotoxic molecules upon target recognition. Cell viability, total CD3+ cell number, proliferative capacity and cytotoxic potential remained stable throughout storage of up to 72 h after end of leukapheresis. Conclusion: Clinical-grade SARS-CoV-2-specific T cells are functional, have proliferative capacity and target-specific cytotoxic potential. Their function and phenotype remain stable for several days after enrichment. The adoptive transfer of partially matched, viable human SARS-CoV-2-specific T lymphocytes collected from convalescent individuals may provide the opportunity to support the immune system of COVID-19 patients at risk for severe disease.

Interferon Gamma Secretion of Adaptive and Innate Immune Cells as a Parameter to Describe Leukaemia-Derived Dendritic-Cell-Mediated Immune Responses in Acute Myeloid Leukaemia in vitro.

INTRODUCTION: Myeloid leukaemic blasts can be converted into leukaemia-derived dendritic cells (DCleu), characterised by the simultaneous expression of dendritic- and leukaemia-associated antigens, which have the competence to prime and enhance (leukaemia-specific) immune responses with the whole leukaemic antigen repertoire. To display and further specify dendritic cell (DC)- and DCleu-mediated immune responses, we analysed the interferon gamma (IFNy) secretion of innate and adaptive immune cells. METHODS: DC/DCleu were generated from leukaemic whole blood (WB) with (blast)modulatory Kit-I (granulocyte-macrophage colony-stimulating factor [GM-CSF] + Picibanil [OK-432]) and Kit-M (GM-CSF + prostaglandin E1) and were used to stimulate T cell-enriched immunoreactive cells. Initiated anti-leukaemic cytotoxicity was investigated with a cytotoxicity fluorolysis assay. Initiated IFNy secretion of T, NK, CIK, and iNKT cells was investigated with a cytokine secretion assay (CSA). IFNy positivity was additionally evaluated with an intracellular cytokine assay (ICA). Recent activation of leukaemia-specific cells was verified through addition of leukaemia-associated antigens (LAA; WT-1 and Prame). RESULTS: We found Kit-I and Kit-M competent to generate mature DC and DCleu from leukaemic WB without induction of blast proliferation. Stimulation of immunoreactive cells with DC/DCleu regularly resulted in an increased anti-leukaemic cytotoxicity and increased IFNy secretion of T, NK, and CIK cells, pointing to the significant role of DC/DCleu in leukaemia-specific alongside anti-leukaemic reactions. Interestingly, an addition of LAA did not further increase IFNy secretion, suggesting an efficient activation of leukaemia-specific cells. Here, both the CSA and ICA yielded comparable frequencies of IFNy-positive cells. Remarkably, the anti-leukaemic cytotoxicity positively correlated with the IFNy secretion in TCD3+, TCD4+, TCD8+, and NKCD56+ cells. CONCLUSION: Ultimately, the IFNy secretion of innate and adaptive immune cells appeared to be a suitable parameter to assess and monitor the efficacy of in vitro and potentially in vivo acute myeloid leukaemia immunotherapy. The CSA in this regard proved to be a convenient and reproducible technique to detect and phenotypically characterise IFNy-secreting cells. In respect to our studies on DC-based immunomodulation, we were able to display the potential of DC/DCleu to induce or improve leukaemia-specific and anti-leukaemic activity.

Variances in Antiviral Memory T-Cell Repertoire of CD45RA- and CD62L-Depleted Lymphocyte Products Reflect the Need of Individual T-Cell Selection Strategies to Reduce the Risk of GvHD while Preserving Antiviral Immunity in Adoptive T-Cell Therapy.

INTRODUCTION: Viral infections and reactivations still remain a cause of morbidity and mortality after hematopoietic stem cell transplantation due to immunodeficiency and immunosuppression. Transfer of unmanipulated donor-derived lymphocytes (DLI) represents a promising strategy for improving cellular immunity but carries the risk of graft versus host disease (GvHD). Depleting alloreactive naïve T cells (TN) from DLIs was implemented to reduce the risk of GvHD induction while preserving antiviral memory T-cell activity. Here, we compared two TN depletion strategies via CD45RA and CD62L expression and investigated the presence of antiviral memory T cells against human adenovirus (AdV) and Epstein-Barr virus (EBV) in the depleted fractions in relation to their functional and immunophenotypic characteristics. METHODS: T-cell responses against ppEBV_EBNA1, ppEBV_Consensus and ppAdV_Hexon within TN-depleted (CD45RA-/CD62L-) and TN-enriched (CD45RA+/CD62L+) fractions were quantified by interferon-gamma (IFN-γ) ELISpot assay after short- and long-term in vitro stimulation. T-cell frequencies and immunophenotypic composition were assessed in all fractions by flow cytometry. Moreover, alloimmune T-cell responses were evaluated by mixed lymphocyte reaction. RESULTS: According to differences in the phenotype composition, antigen-specific T-cell responses in CD45RA- fraction were up to 2 times higher than those in the CD62L- fraction, with the highest increase (up to 4-fold) observed after 7 days for ppEBV_EBNA1-specific T cells. The CD4+ effector memory T cells (TEM) were mainly responsible for EBV_EBNA1- and AdV_Hexon-specific T-cell responses, whereas the main functionally active T cells against ppEBV_Consensus were CD8+ central memory T cells (TCM) and TEM. Moreover, comparison of both depletion strategies indicated that alloreactivity in CD45RA- was lower than that in CD62L- fraction. CONCLUSION: Taken together, our results indicate that CD45RA depletion is a more suitable strategy for generating TN-depleted products consisting of memory T cells against ppEBV_EBNA1 and ppAdV_Hexon than CD62L in terms of depletion effectiveness, T-cell functionality and alloreactivity. To maximally exploit the beneficial effects mediated by antiviral memory T cells in TN-depleted products, depletion methods should be selected individually according to phenotype composition and CD4/CD8 antigen restriction. TN-depleted DLIs may improve the clinical outcome in terms of infections, GvHD, and disease relapse if selection of pathogen-specific donor T cells is not available.