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Despite routine post-transplant viral monitoring and pre-emptive therapy, viral infections remain a major cause of allogeneic hematopoietic cell transplantation-related morbidity and mortality.We here aimed to prospectively assess the kinetics and the magnitude of cytomegalovirus-(CMV), Epstein Barr virus-(EBV), and BK virus-(BKV)-specific T cell responses post-transplant and evaluate their role in guiding therapeutic decisions by patient risk-stratification.The tri-virus-specific immune recovery was assessed by Elispot, in 50 consecutively transplanted patients, on days +20, +30, +60, +100, +150, +200 post-transplant and in case of reactivation, weekly for 1 month.The great majority of the patients experienced at least one reactivation, while over 40% of them developed multiple reactivations from more than one of the tested viruses, especially those transplanted from matched or mismatched unrelated donors. The early reconstitution of virus-specific immunity (day +20), favorably correlated with transplant outcomes. Εxpanding levels of CMV-, EBV-, and BKV-specific T cells (VSTs) post-reactivation coincided with decreasing viral load and control of infection. Certain cut-offs of absolute VST numbers or net VST cell expansion post-reactivation were determined, above which, patients with CMV or BKV reactivation had 90% probability of complete response (CR).Immune monitoring of virus-specific T-cell reconstitution post-transplant may allow risk-stratification of virus reactivating patients and enable patient-tailored treatment. The identification of individuals with high probability of CR will minimize unnecessary overtreatment and drug-associated toxicity while allowing candidates for pre-emptive intervention with adoptive transfer of VSTs to be appropriately selected.

Advances in immunotherapy with T cells armed with chimeric antigen receptors (CAR-Ts), opened up new horizons for the treatment of B-cell lymphoid malignancies. However, the lack of appropriate targetable antigens on the malignant myeloid cell deprives patients with refractory acute myeloid leukaemia of effective CAR-T therapies. Although non-engineered T cells targeting multiple leukaemia-associated antigens [i.e. leukaemia-specific T cells (Leuk-STs)] represent an alternative approach, the prerequisite challenge to obtain high numbers of dendritic cells (DCs) for large-scale Leuk-ST generation, limits their clinical implementation. We explored the feasibility of generating bivalent-Leuk-STs directed against Wilms tumour 1 (WT1) and preferentially expressed antigen in melanoma (PRAME) from umbilical cord blood units (UCBUs) disqualified for allogeneic haematopoietic stem cell transplantation. By repurposing non-transplantable UCBUs and optimising culture conditions, we consistently produced at clinical scale, both cluster of differentiation (CD)34+ cell-derived myeloid DCs and subsequently polyclonal bivalent-Leuk-STs. Those bivalent-Leuk-STs contained CD8+ and CD4+ T cell subsets predominantly of effector memory phenotype and presented high specificity and cytotoxicity against both WT1 and PRAME. In the present study, we provide a paradigm of circular economy by repurposing unusable UCBUs and a platform for future banking of Leuk-STs, as a 'third-party', 'off-the-shelf' T-cell product for the treatment of acute leukaemias.

Background The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic poses an urgent need for the development of effective therapies for coronavirus disease 2019 (COVID-19). Methods We first tested SARS-CoV-2–specific T-cell (CοV-2-ST) immunity and expansion in unexposed donors, COVID-19–infected individuals (convalescent), asymptomatic polymerase chain reaction (PCR)–positive subjects, vaccinated individuals, non–intensive care unit (ICU) hospitalized patients, and ICU patients who either recovered and were discharged (ICU recovered) or had a prolonged stay and/or died (ICU critical). CoV-2-STs were generated from all types of donors and underwent phenotypic and functional assessment. Results We demonstrate causal relationship between the expansion of endogenous CoV-2-STs and the disease outcome; insufficient expansion of circulating CoV-2-STs identified hospitalized patients at high risk for an adverse outcome. CoV-2-STs with a similarly functional and non-alloreactive, albeit highly cytotoxic, profile against SARS-CoV-2 could be expanded from both convalescent and vaccinated donors generating clinical-scale, SARS-CoV-2–specific T-cell products with functional activity against both the unmutated virus and its B.1.1.7 and B.1.351 variants. In contrast, critical COVID-19 patient-originating CoV-2-STs failed to expand, recapitulating the in vivo failure of CoV-2–specific T-cell immunity to control the infection. CoV-2-STs generated from asymptomatic PCR-positive individuals presented only weak responses, whereas their counterparts originating from exposed to other seasonal coronaviruses subjects failed to kill the virus, thus disempowering the hypothesis of protective cross-immunity. Conclusions Overall, we provide evidence on risk stratification of hospitalized COVID-19 patients and the feasibility of generating powerful CoV-2-ST products from both convalescent and vaccinated donors as an “off-the shelf” T-cell immunotherapy for high-risk patients.

Invasive aspergillosis (IA) represents a leading cause of mortality in immunocompromised patients. Although adoptive immunotherapy with Aspergillus-specific T cells (Asp-STs) represents a promising therapeutic approach against IA, the complex and costly production limits its broader application. We generated Asp-STs from a single blood draw of healthy individuals or IA patients in only 10 days, by either Aspergillus fumigatus (AF) lysate or peptide stimulation of mononuclear cells. The cells were phenotypically and functionally characterized, and safety was assessed in xenografts. Healthy donor-derived and lysate- or peptide-pulsed Asp-STs presented comparable fold expansion, immunophenotype, and Th1 responses. Upon cross-stimulation, only the lysate-pulsed Asp-STs were empowered to respond to peptide stimulation, although both cell products induced hyphal damage. Importantly, Asp-STs cross-reacted with other fungal species and did not induce alloreactivity in vivo. IA patient-derived T cells displayed an anergic phenotype that prohibited sufficient expansion and yield of meaningful doses of Asp-STs for autologous immunotherapy. Using a rapid and simple process, we generated, from healthy donors but not IA patients, functionally active Asp-STs of broad specificity and at clinically relevant numbers. Such an approach may form the basis for the effective management of IA in the context of allogeneic hematopoietic cell transplantation.

Adoptive immunotherapy (AI) with pathogen-specific T cells is a promising alternative to pharmacotherapy for the treatment of opportunistic infections after allogeneic hematopoietic cell transplantation or solid organ transplantation. However, clinical implementation of AI is limited to patients not receiving high-dose steroids, a prerequisite for optimal T-cell function, practically excluding the most susceptible to infections patients from the benefits of AI. To address this issue, we here rapidly generated, clinical doses of a steroid-resistant T-cell product, simultaneously targeting four viruses (adenovirus, cytomegalovirus, Epstein Barr virus, and BK virus) and the fungus Aspergillus fumigatus, by genetic disruption of the glucocorticoid receptor (GR) gene using CRISPR/CAS9 ribonucleoprotein delivery. The product, “Cerberus” T cells (Cb-STs), was called after the monstrous three-headed dog of Greek mythology, due to its triple potential; specificity against viruses, specificity against fungi and resistance to glucocorticoids. Following efficient on-target GR disruption and minimal off-target editing, the generated Cb-STs maintained the characteristics of pentavalent-STs, their unedited counterparts, including polyclonality, memory immunophenotype, specificity, and cytotoxicity while they presented functional resistance to dexamethasone. Cb-STs may become a powerful, one-time treatment for severely immunosuppressed patients under glucocorticoids who suffer from multiple, life-threatening infections post-transplant, and for whom therapeutic choices are limited.

Viral infections, mainly by cytomegalovirus (CMV), Epstein Barr virus (EBV) and polyomavirus type I (BKV), are major causes of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). As effective immune responses against human viruses rely on an armamentarium of T-cell receptor (TR) repertoire capable of recognizing a broad range of antigenic peptides of those pathogens, reconstitution of antiviral immunity, either by spontaneous generation of endogenous virus-specific T cells (VSTs) or by adoptive immunotherapy with VSTs, plays a critical role to fight infections. We here evaluated the diversity and clonality of TR repertoire of functional tri-virus-specific T cell products generated from immunocompetent donors (n=10) and compared their TR gene repertoire to that of peripheral blood mononuclear cells (PBMCs) from patients who had undergone allo-HSCT (n=5). To generate tri-VSTs, PBMCs derived from 15-20ml of peripheral blood of normal donors, were exposed to EBV, CMV and BKV overlapping peptides and cultured in the presence of interleukin 4 (IL-4) and IL-7 for 10 days in G-rex bioreactors. Specificity of donor-derived VSTs and patient-derived PBMCs was measured by IFN-γElispot. TR diversity was investigated by next-generation sequencing on a MiSeq Sequencer, after amplification of TR beta chain gene rearrangements by RT-PCR with the BIOMED-2 protocol. Raw NGS reads were filtered based on their length and quality and the filtered-in sequences were submitted to IMGT/HighVQUEST. Metadata analysis and clonotype computation were performed using a validated in-house bioinformatics platform. As clonotype we defined sequences carrying the same TRBV gene and identical CDR3 amino acid sequence. Tri-VSTs provided 947,298 productive TRBV-TRBD-TRBJ rearrangements and a polyclonal and highly diverse TR gene repertoire, consisting of a total of 169,502 unique clonotypes (average: 16,950/sample, range 4,057-45,602), 64,971 (38.3%) of which were expanded (corresponding to more than one sequence). In terms of clonality, the mean relative frequency of the major clonotype in all tri-VSTs was 12.6% (range 3.3-29.2%). Interestingly, among tri-VST cell lines, 637 clonotypes were shared (present in >2/10 samples), 80 were highly shared (present in >3/10 samples) while 7 were present in 6-8 different VST lines and largely expanded, accounting for up to 29.2% of all sequences. Importantly, there were 65 of 96 major VST clonotypes shared, thus suggesting that they were potentially associated with recognition of the targeted viruses. Given that 4/10 VSTs cell lines were not specific for CMV, while being EBV-and BKV-specific, dominant TRs in those 4 cell lines can potentially be associated with EBV- or BKV-activity. By searching a public database of TR clonotypes with known reactivity against EBV and/or CMV (ShugayM, Nucleic Acids Research, 2018), we found 8 shared EBV-specific and 4 shared CMV-specific clonotypes among our VSTs and the 499 public clonotypes. When we compared the produced VSTs with PBMCs from 3 allo-grafted patients with circulating CMV-, BKV- and EBV-specific T cells and previous viral reactivation, we detected 163 shared clonotypes. Likewise, we observed 21 and 23 shared clonotypes in similar frequencies, between VSTs and PBMCs from 2 patients with CMV- or BKV-specific T cell immunity. These data identify clones that potentially expand in vivo and protect patients from viral infections. Overall, our findings reveal high levels of TR clonality in cell lines enriched for T cells reactive against EBV and/or CMV and/or BKV and provide insights into the TR repertoire of ex vivo- or endogenously-generated VSTs. Our approach may help to identify optimal TRs for immunotherapy as well as TRs which can be used as a tool for risk stratification of viral infections. Disclosures Agathangelidis: Gilead: Research Funding. Gemenetzi:Gilead: Research Funding. Stamatopoulos:Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding. Hadzidimitriou:Gilead: Research Funding; Abbvie: Research Funding; Janssen: Honoraria, Research Funding.