key: cord-0749971-zf3cs82n authors: Guerreiro, Manuel; Aguilar‐Gallardo, Cristóbal; Montoro, Juan; Francés‐Gómez, Clara; Latorre, Víctor; Luna, Irene; Planelles, Dolores; Carrasco, María Paz; Gómez, María Dolores; González‐Barberá, Eva María; Aguado, Cristina; Sempere, Amparo; Solves, Pilar; Gómez‐Seguí, Inés; Balaguer‐Rosello, Aitana; Louro, Alberto; Perla, Aurora; Larrea, Luis; Sanz, Jaime; Arbona, Cristina; de la Rubia, Javier; Geller, Ron; Sanz, Miguel Ángel; Sanz, Guillermo; Luis Piñana, José title: Adoptive transfer of ex vivo expanded SARS‐CoV‐2‐specific cytotoxic lymphocytes: A viable strategy for COVID‐19 immunosuppressed patients? date: 2021-03-31 journal: Transpl Infect Dis DOI: 10.1111/tid.13602 sha: 92b362961fdf8c2e051decb3f0a5ae21fd8d3adf doc_id: 749971 cord_uid: zf3cs82n Cellular and humoral response to acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infections is on focus of research. We evaluate herein the feasibility of expanding virus‐specific T cells (VST) against SARS‐CoV‐2 ex vivo through a standard protocol proven effective for other viruses. The experiment was performed in three different donors' scenarios: (a) SARS‐CoV‐2 asymptomatic infection/negative serology, (b) SARS‐CoV‐2 symptomatic infection/positive serology, and (c) no history of SARS‐CoV‐2 infection/negative serology. We were able to obtain an expanded VST product from donors 1 and 2 (1.6x and 1.8x increase of baseline VST count, respectively) consisting in CD3 + cells (80.3% and 62.7%, respectively) with CD4 + dominance (60% in both donors). Higher numbers of VST were obtained from the donor 2 as compared to donor 1. T‐cell clonality test showed oligoclonal reproducible peaks on a polyclonal background for both donors. In contrast, VST could be neither expanded nor primed in a donor without evidence of prior infection. This proof‐of‐concept study supports the feasibility of expanding ex vivo SARS‐CoV‐2‐specific VST from blood of convalescent donors. The results raise the question of whether the selection of seropositive donors may be a strategy to obtain cell lines enriched in their SARS‐CoV‐2‐specificity for future adoptive transfer to immunosuppressed patients. The coronavirus disease 2019 (COVID-19) pandemic is causing an enormous health and economic global impact worldwide. Solid tumor or oncohematological patients who develop COVID-19 are at higher risk of mortality, 1,2 surely due to their hampered humoral and cellular immune response against SARS-CoV-2. 3, 4 Adaptive T cell immune responses are increasingly recognized as key factor controlling viral clearance, severity of COVID-19, and humoral immune responses. 5 A deep understanding of T cell responses to SARS-CoV-2 is critical to improve our assessment of the probability of SARS-CoV-2 reinfection, COVID-19 prognosis, the estimation of general population immunity, as well as for guiding vaccine development. Although immunoglobulin G (IgG) and M (IgM) seroconversion rate after COVID-19 is common, it can vary from 80% to 100% in symptomatic and 15% to 95% in asymptomatic patients after SARS-CoV-2 infection. [6] [7] [8] [9] [10] Moreover, the humoral response is not always achieved, and the presence of IgG can be as short as few days or weeks after recovery in nearly 40% of patients. 6 In contrast, strong and long-lasting CD4 and CD8 T cell responses 5, 11 have been observed in nearly all infected cases, highlighting their predominant role in SARS-CoV-2 control and clearance. Currently, there are several ways to asses T cell response to SARS-CoV-2 infection such as ex vivo T cell flow cytometry and ELISPOTbased assays. 11, 12 However, limitations of these tests include expertise in interpretation, lack of consensus in the standardization of the methodology, and low burden of circulating SARS-CoV-2-specific T cells (<0.1% of total lymphocyte subset in peripheral blood). 12 Furthermore, T cell cross-reactivity observed with other common seasonal human coronavirus could be regarded as a limitation since it may overestimate the real immunity in the general population. 5, 13 This issue is further aggravated by the current lack of evidence that such cross-reactivity could confer functional protection against SARS-CoV-2. 14 Innovative approaches, such as ex vivo virus-specific T cells (VST) expansion used for cytomegalovirus (CMV) and Epstein Barr virus (EBV), 15, 16 may be of value for SARS-CoV-2 since it could offer a great number of VST against SARS-CoV-2 for clinical or laboratory research purposes. 17 We present herein a proof-of-concept experiment to investigate the feasibility of expanding SARS-CoV-2 VST ex vivo. We report the results of extending the applicability of optimized VST expansion protocol against CMV and EBV to the SARS-CoV-2 and discuss about the potential implications of our findings. For the study purpose, we selected two healthcare workers with PCR-confirmed SARS-CoV-2 infection on 11 and 26 March 2020, respectively, and a volunteer healthy donor. All three volunteer donors were previously registered in REDOCEL, a database with highresolution typed blood donors Human Leukocyte Antigen (HLA) system consenting to be contacted whether cellular product donations were required for adoptive therapies. This donor bank is enriched with young individuals carrying common Spanish HLA Class I/ II alleles, which facilitates HLA matching for future patients. To carry out our proof-of-concept experiments, donors were selected in order to included three different immunologic profiles: two donors with PCR-confirmed history of SARS-CoV-2 infection, one of which was asymptomatic with no seroconversion (donor 1), while the other was mildly symptomatic and had detectable circulating IgG and IgM antibodies (donor 2), and a third donor without previous history of infection or seroconversion (donor 3). Peripheral blood mononuclear cells (PBMCs) were obtained from 50 mL blood donations on 12 May 2020 after written informed consent. Cell processing and ex vivo expansion experiments were conducted at the GMP facility in Hospital Universitario y Politécnico La Fe. Summary of the entire protocol applied is described in Figure 1 . This project has been approved by the ethical committee and institutional review board (registration number 2020-123-1). HLA-A, -B, -C, -DRB1, and -DQB1 loci typing of the blood donors included in the experiments was performed in the Histocompatibility Laboratory of the Valencia Transfusion Center by next generation sequencing (NGS) using commercially available reagents (GenDX, Utrecht, The Netherlands) and a MiniSeq platform (Illumina). SARS-CoV-2 diagnostic was performed by real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) by detection from blood of convalescent donors. The results raise the question of whether the selection of seropositive donors may be a strategy to obtain cell lines enriched in their SARS-CoV-2-specificity for future adoptive transfer to immunosuppressed patients. The analytical panel designed to evaluate the hematological changes related to SARS-CoV-2 infection included absolute lymphocyte and platelet counts, lymphocyte subpopulations analysis, as well as the serum levels of C-reactive protein, lactate dehydrogenase, D-dimers, fibrinogen, ferritin, cardiac troponin, and IL-6. Serum C-reactive protein (CRP) and Ferritin were measured by an immunoturbidimetric assay (Roche Hitachi), IL-6, and Procalcitonin by "ECLIA" method and LDH activity with the IFCC reference procedure. In the donors with PCR confirmed COVID-19 history, the neutralization capacity of circulating antibodies against the spike protein of SARS-CoV-2 was assessed using a vesicular stomatitis virus pseudotyped with the SARS-CoV-2 Spike protein. Experiments were performed as previously described 18 with the exception that all tests were done in triplicate using fourfold antibody dilutions ranging from 1:20 to 1:20,480. The dose resulting in 50% neutralization was calculated using a three parameter logistic regression with the drc package in R using the LL3 function. Donor-derived dendritic cells (DCs) were generated from freshly PBMCs isolated by gradient centrifugation with Ficoll-Paque (Lymphoprep StemCell Technologies) from 50 mL of anticoagulated (acid-citrate-dextrose) venous blood. After separating the F I G U R E 1 Expansion protocol. Schematic representation of the 31-day expansion process of SARS-CoV-2 VST. The protocol starts with a density gradient separation of the blood sample donation to obtain peripheral mononuclear cells (PBMCs) that will be magnetically separated into CD14 positive and negative fractions. The CD14 positively selected monocytes are plated in petri dishes and stimulated with a cocktail of cytokines for a period of 10 days in order to differentiate them into DCs. From the CD14 negative fraction, a portion is cultivated in T75 flasks for 14 days in the presence phytohemagglutinin-P (PHA) to induce lymphocyte blasts (PHA-blasts), followed a 30 Gy gamma-irradiation and cryopreservation until their usage as antigen presenting cells, while the rest of the negative fraction is cryopreserved until the start of the T cell culture. Once DCs are differentiated, this portion of the negative fraction is thawed and seeded on G-Rex for an initial stimulation with SARS-CoV-2-peptide loaded DCs (day 0), followed by 2 restimulations using peptide-loaded PHA-Blasts (day 7 and 14). On the last day of expansion (day 21), the culture is sampled for characterization and cells are harvested, aliquoted, cryopreserved, and stored in liquid nitrogen until their use for adoptive transfer into HLA-matched severe COVID-19 patients PBMCs, monocytes were isolated by immunomagnetic bead positive selection using the CliniMACS CD14 Reagent (Miltenyi Biotec, Germany) according to the manufacturer's instructions. At this point, 50% of the CD14 negative fraction was cryopreserved with 10% DMSO + 90% human AB serum in polystyrene cryovials and kept at -80°C until its further usage. The CD14 + selected monocytes were matured for 10 days using a cocktail of cytokines into monocyte-derived DCs. Briefly, cells were seeded in petri dishes at 37°C and 5% CO 2 in IMDM (Lonza Walkersville, USA) in a concentration of 1 × 10 6 cells/mL and differen- The noncryopreserved CD14 negative fraction was used to generate PHA blasts required for repeated cycles of culture restimulation. Briefly, cells were culture for 7 days in T75 cm 2 flasks at concentration of 1 × 10 6 cells/mL in RPMI 1640 (Sigma) supplemented with After the DCs differentiation, the previously cryopreserved CD14 negative fraction was thawed and cells seeded in a gas-permeable Sterility of the expanded cells was assessed at the end of the culture by direct Gram staining, by testing for aerobic/anaerobic bacteria contamination using BacT/ALERT FA/FN Plus detection media (Biomerieux Diagnostics, USA) or fungal/mycobacterial contamination using BACTEC MYCO/F LYTIC (BD) medium and mycoplasma contamination was also ruled out by qPCR. Expanded cells were aliquoted in polystyrene cryovials and cryopreserved with 10% DMSO + 90% human AB serum using a passive freezing container (Mr Frosty, Thermo Fischer Scientific) before being transfer into a liquid nitrogen tank. Clinical characteristics of each donor are detailed in Table 1 T cell expansion and cell cultures were analyzed for cell number growth, cellular content, specificity according to cytokine production (day 14), and cytotoxic potential (day 21), as detailed in Table 2 . In terms of cellular growth, we observed an expected decrease in antigen-specific T cells. The intensity of these peaks was more prominent for donor 2, which is also in accordance with the higher percentage of SARS-CoV-2 VSTs observed by flowcytometry (Figure 4 ). This proof-of-concept experiment shows that VST expansion against SARS-CoV-2 is feasible under GMP conditions and can achieve a nearly twofold increase in the number of this VST from patients with symptomatic and asymptomatic PCR-confirmed SARS-CoV-2 infection. We observed a predominance of CD4 + T cells in the final VST product. The SARS-CoV-2 specificity of the expanded product was Adaptive NK cells could also play a protective role against this new emerging virus. 24, 25 Different approaches exist to obtain VST. One consists of isolating these T-cells from whole blood or leukapheresis products with virus reactive cells using an automated device capturing IFNγ-secreting cells. This approach has been already assayed using In this proof-of-concept study, we conclude that it is possible to expand ex vivo SARS-CoV-2 VSTs from convalescent donors of F I G U R E 4 Clonality study. T cell clonality assessment via multiplexed amplification of the TCR γ locus in ex vivo blood samples (day 0) and in the final expanded cellular product (day 21) for both donor 1 and donor 2. Multiple reproducible peaks/bands could be found in donor 1 (seronegative and in which expansion of SARS-Cov2-specific was markedly inferior), suggesting the presence of multiple clones. For donor 2 (seropositive in which an effective expansion was observed), multiple reproducible peaks/bands were observed, suggesting an oligoclonal product SARS-CoV-2 infection, using a standard protocol to obtain virusspecific VSTs. Contrariwise, this approach was not successful to prime de novo responses to SARS-CoV-2 peptides in an uninfected healthy donor. The author(s) declare that they have no conflict of interests. The study was conducted according to the guidelines of the Fe (protocol code: 2020-123-1 and date of approval: March 27, 2020) Informed consent was obtained from all subjects involved in the study. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. 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