Extracorporeal therapy of sepsis by purified granulocyte concentrates: ex vivo circulation model.

BACKGROUND: Immune cell dysfunction is a central part of immune paralysis in sepsis. Granulocyte concentrate (GC) transfusions can induce tissue damage via local effects of neutrophils. The hypothesis of an extracorporeal plasma treatment with granulocytes is to show beneficial effects with fewer side effects. Clinical trials with standard GC have supported this approach. This ex vivo study investigated the functional properties of purified granulocyte preparations during the extracorporeal plasma treatment. METHODS: Purified GC were stored for up to 3 days and compared with standard GC in an immune cell perfusion therapy model. The therapy consists of a plasma separation device and an extracorporeal circuit. Plasma is perfused through the tubing system with donor immune cells of the GC, and only the treated plasma is filtered for re-transfusion. The donor immune cells are retained in the extracorporeal system and discarded after treatment. Efficacy of granulocytes regarding phagocytosis, oxidative burst as well as cell viability and metabolic parameters were assessed. RESULTS: In pGC, the metabolic surrogate parameters of cell functionality showed comparable courses even after a storage period of 72 h. In particular, glucose and oxygen consumption were lower after extended storage. The course of lactate dehydrogenase concentration yields no indication of cell impairment in the extracorporeal circulation. The cells were viable throughout the entire study period and exhibited preserved phagocytosis and oxidative burst functionality. CONCLUSION: The granulocytes demonstrated full functionality in the 6 h extracorporeal circuits after 3 days storage and in septic shock plasma. This is demonstrating the functionality of the system and encourages further clinical studies.

Risk of transfusion-transmitted hepatitis E virus infection from pool-tested platelets and plasma.

BACKGROUND AND AIMS: Immunocompromised patients are at risk of chronic hepatitis E which can be acquired by blood transfusions. Currently, screening of blood donors (BDs) for HEV RNA with a limit of detection (LOD) of 2,000 IU/ml is required in Germany. However, this may result in up to 440,000 IU of HEV RNA in blood products depending on their plasma volume. We studied the residual risk of transfusion-transmitted (tt) HEV infection when an LOD of 2,000 IU/ml is applied. METHODS: Highly sensitive individual donor testing for HEV RNA on the Grifols Procleix Panther system (LOD 7.89 IU/ml) was performed. HEV loads were quantified by real-time PCR. RESULTS: Of 16,236 donors, 31 (0.19%) were HEV RNA positive. Three BDs had viral loads between 710 and 2,000 IU/ml, which pose a significant risk of tt hepatitis E with any type of blood product. Eight BDs had viral loads of >32 to 710 IU/ml, which pose a risk of tt hepatitis E with platelet or plasma transfusions because of their higher plasma volume compared to red blood cell concentrates. Eight of these 11 potentially infectious BDs were seronegative for HEV, indicating a recent infection. Only 8 of 31 donors had viral loads >2,000 IU/ml that would also have been detected by the required screening procedure and 12 had very low HEV loads (<32 IU/ml). CONCLUSIONS: Screening of BDs with an LOD of 2,000 IU/ml reduced the risk of tt HEV infection by about 73% for red blood cell concentrates but by just 42% for platelet and fresh frozen plasma transfusions. Single donor screening (LOD <32 IU/ml) should lead to an almost 100% risk reduction. LAY SUMMARY: Immunocompromised patients, such as solid organ or hematopoietic stem cell recipients, are at risk of chronic hepatitis E, which can be acquired via blood transfusions. The risk of transfusion-transmitted hepatitis E in these patients may not be sufficiently controlled by (mini-)pool hepatitis E virus RNA screening of blood donors. Single donor screening should be considered to improve the safety of blood products.

Antiviral T-Cell Frequencies in a Healthy Population: Reference Values for Evaluating Antiviral Immune Cell Profiles in Immunocompromised Patients.

Viral infections and reactivations are major causes of morbidity and mortality after hematopoietic stem cell (HSCT) and solid organ transplantation (SOT) as well as in patients with immunodeficiencies. Latent herpesviruses (e.g., cytomegalovirus, Epstein-Barr virus, and human herpesvirus 6), lytic viruses (e.g., adenovirus), and polyomaviruses (e.g., BK virus, JC virus) can cause severe complications. Antiviral drugs form the mainstay of treatment for viral infections and reactivations after transplantation, but they have side effects and cannot achieve complete viral clearance without prior reconstitution of functional antiviral T-cell immunity. The aim of this study was to establish normal ranges for virus-specific T-cell (VST) frequencies in healthy donors. Such data are needed for better interpretation of VST frequencies observed in immunocompromised patients. Therefore, we measured the frequencies of VSTs against 23 viral protein-derived peptide pools from 11 clinically relevant human viruses in blood from healthy donors (n = 151). Specifically, we determined the VST frequencies by interferon-gamma enzyme-linked immunospot assay and classified their distribution according to age and gender to allow for a more specific evaluation and prediction of antiviral immune responses. The reference values established here provide an invaluable tool for immune response evaluation, intensity of therapeutic drugs and treatment decision-making in immunosuppressed patients. This data should make an important contribution to improving the assessment of immune responses in immunocompromised patients.

Prolonged storage of purified granulocyte concentrates: Introduction of a new purification method.

BACKGROUND: Use of donor granulocyte concentrate (GC) has been limited due to its short storage time of 6-24 h, which is partially due to residual red blood cells (RBCs) and platelets and the resulting lactate production leading to an acidotic milieu. To increase this storage time, we developed a closed system procedure compatible with standard blood bank technologies to remove RBC and platelets and to enrich the GC. METHODS: Standard GCs (sGCs) were sedimented, washed twice with 0.9% sodium chloride (NaCl), and resuspended in blood group-identical fresh frozen plasma. The resulting purified GCs (pGCs) were then stored in platelet bags at a cell concentration of about 5 × 107  ± 1.8 × 107 leukocytes/ml without agitation at room temperature for up to 72 h. Cell count and viability, pH, blood gases, phagocytosis, and oxidative burst were monitored daily. RESULTS: A significant reduction in RBC (98%) through sedimentation, and platelets (96%) by washing, purified the white blood cell (WBC) population and enriched the granulocytes to 96% of the WBC in the pGC. After 72 h of storage, over 90% of the initial WBC count of pGC remained, was viable (≥97%), and the granulocytes exhibited a high phagocytosis and oxidative burst functionality, comparable to sGC after 24 h. CONCLUSION: Purification extends the maximum storage period of GC from 24 to 72 h and may therefore improve the availability of GC and its clinical use.

Functionality and Cell Senescence of CD4/ CD8-Selected CD20 CAR T Cells Manufactured Using the Automated CliniMACS Prodigy® Platform.

Clinical studies using autologous CAR T cells have achieved spectacular remissions in refractory CD19+ B cell leukaemia, however some of the patient treatments with CAR T cells failed. Beside the heterogeneity of leukaemia, the distribution and senescence of the autologous cells from heavily pretreated patients might be further reasons for this. We performed six consecutive large-scale manufacturing processes for CD20 CAR T cells from healthy donor leukapheresis using the automated CliniMACS Prodigy® platform. Starting with a CD4/CD8-positive selection, a high purity of a median of 97% T cells with a median 65-fold cell expansion was achieved. Interestingly, the transduction rate was significantly higher for CD4+ compared to CD8+ T cells and reached in a median of 23%. CD20 CAR T cells showed a good specific IFN-γ secretion after cocultivation with CD20+ target cells which correlated with good cytotoxic activity. Most importantly, 3 out of 5 CAR T cell products showed an increase in telomere length during the manufacturing process, while telomere length remained consistent in one and decreased in another process. In conclusion, this shows for the first time that beside heterogeneity among healthy donors, CAR T cell products also differ regarding cell senescence, even for cells manufactured in a standardised automated process.

Selected single-nucleotide polymorphisms in FOXE1, SERPINA5, FTO, EVPL, TICAM1 and SCARB1 are associated with papillary and follicular thyroid cancer risk: replication study in a German population.

Several single-nucleotide polymorphisms (SNPs) have been associated with papillary and follicular thyroid cancer (PTC and FTC, respectively) risk, but few have replicated. After analyzing 17525 tag SNPs in 1129 candidate genes, we found associations with PTC risk in SERPINA5, FTO, HEMGN (near FOXE1) and other genes. Here, we report results from a replication effort in a large independent PTC/FTC case-control study conducted in Germany. We evaluated the best tagging SNPs from our previous PTC study and additionally included SNPs in or near FOXE1 and NKX2-1 genes, known susceptibility loci for thyroid cancer. We genotyped 422 PTC and 130 FTC cases and 752 controls recruited from three German clinical centers. We used polytomous logistic regression to simultaneously estimate PTC and FTC associations for 79 SNPs based on log-additive models. We assessed effect modification by body mass index (BMI), gender and age for all SNPs, and selected SNP by SNP interactions. We confirmed associations with PTC and SNPs in FOXE1/HEMGN, SERPINA5 (rs2069974), FTO (rs8047395), EVPL (rs2071194), TICAM1 (rs8120) and SCARB1 (rs11057820) genes. We found associations with SNPs in FOXE1, SERPINA5, FTO, TICAM1 and HSPA6 and FTC. We found two significant interactions between FTO (rs8047395) and BMI (P = 0.0321) and between TICAM1 (rs8120) and FOXE1 (rs10984377) (P = 0.0006). Besides the known associations with FOXE1 SNPs, we confirmed additional PTC SNP associations reported previously. We also found several new associations with FTC risk and noteworthy interactions. We conclude that multiple variants and host factors might interact in complex ways to increase risk of PTC and FTC.

miR-155 is associated with the leukemogenic potential of the class IV granulocyte colony-stimulating factor receptor in CD34⁺ progenitor cells.

Granulocyte colony-stimulating factor (G-CSF) is a major regulator of granulopoiesis on engagement with the G-CSF receptor (G-CSFR). The truncated, alternatively spliced, class IV G-CSFR (G-CSFRIV) has been associated with defective differentiation and relapse risk in pediatric acute myeloid leukemia (AML) patients. However, the detailed biological properties of G-CSFRIV in human CD34(+) hematopoietic stem and progenitor cells (HSPCs) and the potential leukemogenic mechanism of this receptor remain poorly understood. In the present study, we observed that G-CSFRIV-overexpressing (G-CSFRIV(+)) HSPCs demonstrated an enhanced proliferative and survival capacity on G-CSF stimulation. Cell cycle analyses showed a higher frequency of G-CSFRIV(+) cells in the S and G2/M phase. Also, apoptosis rates were significantly lower in G-CSFRIV(+) HSPCs. These findings were shown to be associated with a sustained Stat5 activation and elevated miR-155 expression. In addition, G-CSF showed to further induce G-CSFRIV and miR-155 expression of peripheral blood mononuclear cells isolated from AML patients. A Stat5 pharmacological inhibitor or ribonucleic acid (RNA) interference-mediated silencing of the expression of miR-155 abrogated the aberrant proliferative capacity of the G-CSFRIV(+) HSPCs. Hence, the dysregulation of Stat5/miR-155 pathway in the G-CSFRIV(+) HSPCs supports their leukemogenic potential. Specific miRNA silencing or the inhibition of Stat5-associated pathways might contribute to preventing the risk of leukemogenesis in G-CSFRIV(+) HSPCs. This study may promote the development of a personalized effective antileukemia therapy, in particular for the patients exhibiting higher expression levels of G-CSFRIV, and further highlights the necessity of pre-screening the patients for G-CSFR isoforms expression patterns before G-CSF administration.

Rapid generation of clinical-grade antiviral T cells: selection of suitable T-cell donors and GMP-compliant manufacturing of antiviral T cells.

BACKGROUND: The adoptive transfer of allogeneic antiviral T lymphocytes derived from seropositive donors can safely and effectively reduce or prevent the clinical manifestation of viral infections or reactivations in immunocompromised recipients after hematopoietic stem cell (HSCT) or solid organ transplantation (SOT). Allogeneic third party T-cell donors offer an alternative option for patients receiving an allogeneic cord blood transplant or a transplant from a virus-seronegative donor and since donor blood is generally not available for solid organ recipients. Therefore we established a registry of potential third-party T-cell donors (allogeneic cell registry, alloCELL) providing detailed data on the assessment of a specific individual memory T-cell repertoire in response to antigens of cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (ADV), and human herpesvirus (HHV) 6. METHODS: To obtain a manufacturing license according to the German Medicinal Products Act, the enrichment of clinical-grade CMV-specific T cells from three healthy CMV-seropositive donors was performed aseptically under GMP conditions using the CliniMACS cytokine capture system (CCS) after restimulation with an overlapping peptide pool of the immunodominant CMVpp65 antigen. Potential T-cell donors were selected from alloCELL and defined as eligible for clinical-grade antiviral T-cell generation if the peripheral fraction of IFN-γ(+) T cells exceeded 0.03% of CD3(+) lymphocytes as determined by IFN-γ cytokine secretion assay. RESULTS: Starting with low concentration of IFN-γ(+) T cells (0.07-1.11%) we achieved 81.2%, 19.2%, and 63.1% IFN-γ(+)CD3(+) T cells (1.42 × 10(6), 0.05 × 10(6), and 1.15 × 10(6)) after enrichment. Using the CMVpp65 peptide pool for restimulation resulted in the activation of more CMV-specific CD8(+) than CD4(+) memory T cells, both of which were effectively enriched to a total of 81.0% CD8(+)IFN-γ(+) and 38.4% CD4(+)IFN-γ(+) T cells. In addition to T cells and NKT cells, all preparations contained acceptably low percentages of contaminating B cells, granulocytes, monocytes, and NK cells. The enriched T-cell products were stable over 72 h with respect to viability and ratio of T lymphocytes. CONCLUSIONS: The generation of antiviral CD4(+) and CD8(+) T cells by CliniMACS CCS can be extended to a broad spectrum of common pathogen-derived peptide pools in single or multiple applications to facilitate and enhance the efficacy of adoptive T-cell immunotherapy.

Desmopressin (DDAVP) improves recruitment of activated platelets to collagen but simultaneously increases platelet endothelial interactions in vitro.

Platelet dysfunction can cause clinically relevant bleeding. Treatment with DDAVP is advocated for this condition. DDAVP increases von Willebrand factor (VWF) on endothelial cells (ECs) and in plasma. VWF could facilitate platelet deposition on subendothelial collagen. VWF also facilitates platelet/EC interactions. Therefore DDAVP could precipitate thromboembolic events. We used a flow chamber model to study in vitro and ex vivo if DDAVP alters recruitment of platelets to EC and collagen. Resting or TRAP-activated platelets and EC were treated individually or simultaneously with 0.4 ng/ml DDAVP. Fluorophor-labeled platelets (10(6)/ml) were resuspended in reconstituted blood and superfused across EC and collagen in an in vitro flow chamber model at arterial shear (320 s(-1)). Adhesion of platelets to the respective surface was recorded fluorescence microscopically and platelet covered area was assessed. TRAP significantly induced adhesiveness of platelets for collagen and EC. DDAVP pretreatment of platelets did not affect adhesiveness of resting or TRAP-activated platelets for collagen or EC. Adhesiveness of resting but not TRAP-activated platelets was induced on DDAVP-treated EC. DDAVP-conditioned EC supernatant contained vWF and significantly increased platelet deposition on collagen. Platelets from patients with clinically suspected platelet dysfunction undergoing aortic valve replacement exhibited decreased platelet deposition on collagen surfaces. In summary, our data confirm that DDAVP can induce release of platelet adhesion promoting factors from EC, which is most likely vWF. DDAVP has no direct effect on platelets. Blood samples from DDAVP-treated patients do not exhibit significantly augmented platelet deposition on collagen ex vivo. This influence of released promoting factors might cause an increase of undesirable interactions of platelets with EC.

CMV-, EBV- and ADV-specific T cell immunity: screening and monitoring of potential third-party donors to improve post-transplantation outcome.

Adoptive immunotherapy with virus-specific T lymphocytes can efficiently reconstitute antiviral immunity against cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (ADV) without causing acute toxicity or increasing the risk of graft-versus-host disease. To gain insight into antiviral T cell repertoires and to identify the most efficient antigens for immunotherapy, the frequencies of CMV-, EBV- and ADV-specific T cells in 204 HLA-typed healthy donors were assessed using viral peptides and peptide pools. Confirmatory testing for CMV serology by Western blot technique revealed 19 of 143 (13%) false-positive results. We observed highly significant individual and overall differences in T cell frequencies against CMV, EBV, and ADV antigens, whereas antigen-specific T cells were detected in 100% of CMV- seropositive donors, 73% of EBV- seropositive donors, and 73% of ADV-seropositive donors. At least 124 (61%) potential T cell donors were identified for each virus. Among the tested antigens, frequencies for CMVpp65 and EBVBZLF1 peptide pools were highest. Short-term in vitro peptide stimulation revealed that a donor response to a certain ADV- and EBV-derived peptide may not be determined without prior stimulation. A modified granzyme B ELISpot was used to detect T cell specificity and alloreactivity. Treatment with allogeneic virus-specific cytotoxic T lymphocytes from seropositive third-party donors may be a feasible therapeutic option for infections following cord-blood stem cell transplantation or hematopoietic stem cell transplantation from virus-seronegative donors.

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).

Short-term and long-term leptin exposure differentially affect human natural killer cell immune functions.

Epidemiological studies have indicated that obesity is associated with a higher risk for certain cancers caused by elevated levels of adipocyte-derived hormones. Leptin, one such hormone produced by adipocytes, is a major regulator of metabolism and has also been shown to modulate immunity. However, its role in regulating human natural killer (NK) cell functions is largely unknown. Here, we show that the leptin receptor (Ob-R) is expressed on 5% of NK cells isolated from blood donors, as measured with flow cytometry, and expression of the signal-transducing long form of the leptin receptor Ob-Rb was confirmed with quantitative PCR. The Ob-R+ subpopulation displayed a lower expression of CD16, a cell surface receptor mediating antibody-dependent activation. Short-term stimulation with leptin increased IFNγ secretion, CD69 activation marker expression, and cytotoxic lysis of tumor cells; this was mediated by an improved conjugate forming between NK cells and tumor cells as well as higher expression of tumor necrosis factor-related apoptosis-inducing ligand. On the contrary, long-term incubation with leptin significantly impaired these NK cell immune functions and decreased cell proliferation. In addition, phosphorylation of Jak-2 after leptin stimulation was reduced in peripheral mononuclear blood cells from obese humans compared with normal-weight controls. NK cells represent an immune cell population that is crucial for an effective antitumor response. Here, we show that long-term exposure to leptin, similarly to the situation in obese individuals with elevated serum leptin levels, significantly impairs integral parts of NK cell immune functions, possibly linking leptin to increased cancer susceptibility in obesity.

Low platelet counts after induction therapy for childhood acute lymphoblastic leukemia are strongly associated with poor early response to treatment as measured by minimal residual disease and are prognostic for treatment outcome.

BACKGROUND: Numerous reports have been published on the association between kinetics of leukemic cells during early treatment of childhood acute lymphoblastic leukemia and therapeutic outcome. In contrast, little is known about the prognostic relevance of normal blood counts in this setting. DESIGN AND METHODS: Normal hematopoiesis during and after induction treatment (days 8, 15 and 33) was correlated with therapeutic outcome in a cohort of 256 children with acute lymphoblastic leukemia treated in one of three consecutive ALL-BFM trials at a single institute. Replication analysis of positive findings was performed in an independent cohort of 475 patients from the ALL-BFM 2000 multicenter trial. RESULTS: A platelet count in the first quartile on treatment day 33 and a neutrophil count above the median on day 8 were significantly associated with treatment outcome, conferring multivariate risk ratios for an event of 3.27 (95% confidence interval 1.60-6.69) and 2.26 (95% confidence interval 1.23-4.29), respectively. Replication analysis confirmed the prognostic effect of platelet count on treatment day 33 and demonstrated a strong association with minimal residual disease-based risk group distribution (P<0.00001). CONCLUSIONS: Platelet counts after induction treatment may improve treatment stratification for patients with childhood acute lymphoblastic leukemia and be of particular interest in non-minimal residual disease-based trials. (ALL-BFM 2000 is registered at: ClinicalTrials.gov: NCT00430118. National Cancer Institute: Protocol ID 68529).

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.

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.

Lenograstim with or without dexamethasone for neutrophil mobilization in healthy donors: short-term kinetics of white blood cells and effects of granulocyte apheresis.

OBJECTIVES: To determine the optimal time schedule for neutrophil collection after single mobilization with glycosylated recombinant granulocyte colony-stimulating factor (G-CSF, lenograstim) with or without dexamethasone (DXM). DONORS AND METHODS: In this prospective randomized trial, 26 healthy volunteers were randomly assigned to a single subcutaneous dose of lenograstim 6 µg/kg plus 8-mg DXM (G-CSF/DXM, n = 13) or placebo (G-CSF/placebo, n = 13). Hematological and biochemical parameters were analyzed before and 12, 15, 18, 21, 24, 27, 29, 36, 48, 60, 72, and 84 h and 7 and 30 days after mobilization. Six G-CSF/DXM subjects underwent standard neutrophil apheresis (NA) 12 and 36 h after mobilization. RESULTS: Polymorphonuclear neutrophil (PMN) counts 12 and 21 h after mobilization were 22.7 (16.6-32.8) × 10(9) /L and 22.4 (18.6-30.6) × 10(9) /L for G-CSF/placebo versus 33.1 (24.2-44.9) × 10(9) /L and 32.5 (17.4-39.6) × 10(9) /L for G-CSF/DXM. This mobilization plateau was followed by slow normalization at 72-84 h. The six NA subjects had median PMN yields of 62 (47-101) × 10(9) and 39 (23-42) × 10(9) per therapeutic unit. After the first apheresis, PMN counts sharply decreased to 21.1 (14.8-26.3) × 10(9) /L and then temporarily recovered to 25.9 (18.9-36.5) × 10(9) /L (P ≤ 0.001) over the next 8 h. CONCLUSIONS: Single doses of lenograstim with or without DXM induced a PMN plateau that lasted 9 h (12-21 h after mobilization), with PMN counts suitable for neutrophil collection. Lenograstim plus DXM made it possible to perform NA twice, 12 and 36 h after mobilization.

Generation of HLA-deficient platelets from hematopoietic progenitor cells.

BACKGROUND: Exposure to allogeneic blood products often leads to the development of human leukocyte antigen (HLA) antibodies. Refractoriness to platelet (PLT) transfusion caused by alloimmunization against HLA Class I antigens constitutes a significant clinical problem. STUDY DESIGN AND METHODS: We developed an RNA interference (RNAi)-based approach to silence the expression of HLA Class I molecules on PLTs derived from CD34+ progenitor cells. A lentiviral-based system was used to express short-hairpin RNA (shRNA) targeting ß2-microglobulin (ß2m) transcripts in CD34+ progenitor cells. Differentiation to PLTs was performed by incubating progenitor cells in the presence of thrombopoietin and interleukin-3. RESULTS: The transduction of RNAi cassettes containing the sequences for shRNAs targeting ß2m caused up to 85% reduction of progenitor cells HLA Class I antigen expression, which was maintained in the culture-derived PLTs. The HLA-deficient PLTs derived from HLA-silenced CD34+ cells proved to be fully functional in in vitro tests when compared to peripheral blood-derived PLTs. CONCLUSIONS: Our data show that in vitro generating HLA Class I-deficient PLTs from hematopoietic progenitor cells prove to be feasible. As malignancy risks associated with insertional mutagenesis are not to be expected in anucleated PLTs, provision of HLA-deficient PLTs from large-scale production units may become reality in the management of patients suffering from PLT transfusion refractoriness.

Reappearance of effector T cells is associated with recovery from COVID-19.

BACKGROUND: Elucidating the role of T cell responses in COVID-19 is of utmost importance to understand the clearance of SARS-CoV-2 infection. METHODS: 30 hospitalized COVID-19 patients and 60 age- and gender-matched healthy controls (HC) participated in this study. We used two comprehensive 11-colour flow cytometric panels conforming to Good Laboratory Practice and approved for clinical diagnostics. FINDINGS: Absolute numbers of lymphocyte subsets were differentially decreased in COVID-19 patients according to clinical severity. In severe disease (SD) patients, all lymphocyte subsets were reduced, whilst in mild disease (MD) NK, NKT and γδ T cells were at the level of HC. Additionally, we provide evidence of T cell activation in MD but not SD, when compared to HC. Follow up samples revealed a marked increase in effector T cells and memory subsets in convalescing but not in non-convalescing patients. INTERPRETATION: Our data suggest that activation and expansion of innate and adaptive lymphocytes play a major role in COVID-19. Additionally, recovery is associated with formation of T cell memory as suggested by the missing formation of effector and central memory T cells in SD but not in MD. Understanding T cell-responses in the context of clinical severity might serve as foundation to overcome the lack of effective anti-viral immune response in severely affected COVID-19 patients and can offer prognostic value as biomarker for disease outcome and control. FUNDING: Funded by State of Lower Saxony grant 14-76,103-184CORONA-11/20 and German Research Foundation, Excellence Strategy - EXC2155"RESIST"-Project ID39087428, and DFG-SFB900/3-Project ID158989968, grants SFB900-B3, SFB900-B8.

Dissecting Epstein-Barr Virus-Specific T-Cell Responses After Allogeneic EBV-Specific T-Cell Transfer for Central Nervous System Posttransplant Lymphoproliferative Disease.

Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disease (PTLD) with central nervous system (CNS) involvement is a severe complication after solid organ transplantation. Standard treatment with reduction of immunosuppression and anti-CD20 antibody application often fails leading to poor outcome. Here, we report the case of an 11-year-old boy with multilocular EBV-positive CNS PTLD 10 years after liver transplantation. Complete remission was achieved by repeated intravenous and intrathecal anti-CD20 antibody rituximab administration combined with intrathecal chemotherapy (methotrexate, cytarabine, prednisone) over a time period of 3 months. Due to the poor prognosis of CNS PTLD and lack of EBV-specific T-cells (EBV-CTLs) in patient's blood, we decided to perform EBV-directed T-cell immunotherapy as a consolidating treatment. The patient received five infusions of allogeneic EBV-CTLs from a 5/10 HLA-matched unrelated third-party donor. No relevant acute toxicity was observed. EBV-CTLs became detectable after first injection and increased during the treatment course. Next-generation sequencing (NGS) TCR-profiling verified the persistence and expansion of donor-derived EBV-specific clones. After two transfers, epitope spreading to unrelated EBV antigens occurred suggesting onset of endogenous T-cell production, which was supported by detection of recipient-derived clones in NGS TCR-profiling. Continuous complete remission was confirmed 27 months after initial diagnosis.