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HIV-specific T cells are necessary for control of HIV-1 replication but are largely insufficient for viral clearance. This is due in part to these cells' recognition of immunodominant but variable regions of the virus, which facilitates viral escape via mutations that do not incur viral fitness costs. HIV-specific T cells targeting conserved viral elements are associated with viral control but are relatively infrequent in people living with HIV (PLWH). The goal of this study was to increase the number of these cells via an ex vivo cell manufacturing approach derived from our clinically-validated HIV-specific expanded T-cell (HXTC) process. Using a nonhuman primate (NHP) model of HIV infection, we sought to determine i) the feasibility of manufacturing ex vivo -expanded virus-specific T cells targeting viral conserved elements (CE, CE-XTCs), ii) the in vivo safety of these products, and iii) the impact of simian/human immunodeficiency virus (SHIV) challenge on their expansion, activity, and function. NHP CE-XTCs expanded up to 10-fold following co-culture with the combination of primary dendritic cells (DCs), PHA blasts pulsed with CE peptides, irradiated GM-K562 feeder cells, and autologous T cells from CE-vaccinated NHP. The resulting CE-XTC products contained high frequencies of CE-specific, polyfunctional T cells. However, consistent with prior studies with human HXTC and these cells' predominant CD8 + effector phenotype, we did not observe significant differences in CE-XTC persistence or SHIV acquisition in two CE-XTC-infused NHP compared to two control NHP. These data support the safety and feasibility of our approach and underscore the need for continued development of CE-XTC and similar cell-based strategies to redirect and increase the potency of cellular virus-specific adaptive immune responses., Competing Interests: Author CB is a scientific cofounder and SAB member of Catamaran Bio and Mana Therapeutics; in addition, she has patent 9,885,021 on HIV-specific T cells licensed to Mana Therapeutics. Author CB also serves on the board of directors of Cabaletta Bio and has stock ownership in Repertoire Immune Medicines and Neximmune. She also served on the DSMB for SOBI. H-PK is a scientific cofounder of Ensoma Bio. Author H-PK is a paid advisor for Ensoma Bio. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Dross, Venkataraman, Patel, Huang, Bollard, Rosati, Pavlakis, Felber, Bar, Shaw, Jerome, Mullins, Kiem, Fuller and Peterson.)

Background: Despite similar rates of infection, adults and children have markedly different morbidity and mortality related to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Compared to adults, children have infrequent severe manifestations of acute infection but are uniquely at risk for the rare and often severe Multisystem Inflammatory Syndrome in Children (MIS-C) following infection. We hypothesized that these differences in presentation are related to differences in the magnitude and/or antigen specificity of SARS-CoV-2-specific T cell (CST) responses between adults and children. We therefore set out to measure the CST response in convalescent adults versus children with and without MIS-C following SARS-CoV-2 infection., Methods: CSTs were expanded from blood collected from convalescent children and adults post SARS-CoV-2 infection and evaluated by intracellular flow cytometry, surface markers, and cytokine production following stimulation with SARS-CoV-2-specific peptides. Presence of serum/plasma antibody to spike and nucleocapsid was measured using the luciferase immunoprecipitation systems (LIPS) assay., Findings: Twenty-six of 27 MIS-C patients, 7 of 8 non-MIS-C convalescent children, and 13 of 14 adults were seropositive for spike and nucleocapsid antibody. CST responses in MIS-C patients were significantly higher than children with uncomplicated SARS-CoV-2 infection, but weaker than CST responses in convalescent adults., Interpretation: Age-related differences in the magnitude of CST responses suggest differing post-infectious immunity to SARS-CoV-2 in children compared to adults post uncomplicated infection. Children with MIS-C have CST responses that are stronger than children with uncomplicated SARS-CoV-2 infection and weaker than convalescent adults, despite near uniform seropositivity., Competing Interests: CB is a co-founder and on the scientific advisory boards for Catamaran Bio and Mana Therapeutics with stock and/or ownership, is on the Board of Directors for Caballeta Bio with stock options and has stock in Neximmune and Repertoire Immune Medicines. MK is on a scientific advisory panel for Enzyvant. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Conway, Lazarski, Field, Jensen-Wachspress, Lang, Kankate, Durkee-Shock, Kinoshita, Suslovic, Webber, Smith, Cohen, Burbelo, Zhang, Teach, Ibeh, Delaney, DeBiasi, Keller and Bollard.)

Human Parainfluenza Virus-3 (HPIV3) causes severe respiratory illness in immunocompromised patients and lacks approved anti-viral therapies. A phase I study of adoptively transferred virus-specific T-cells (VSTs) targeting HPIV3 following bone marrow transplantation is underway (NCT03180216). We sought to identify immunodominant epitopes within HPIV3 Matrix protein and their cross-reactivity against related viral proteins. VSTs were generated from peripheral blood of healthy donors by ex-vivo expansion after stimulation with a 15-mer peptide library encompassing HPIV3 matrix protein. Epitope mapping was performed using IFN-γ ELIspot with combinatorial peptide pools. Flow cytometry was used to characterize products with intracellular cytokine staining. In 10 VST products tested, we discovered 12 novel immunodominant epitopes. All products recognized an epitope at the C-terminus. On IFN-γ ELISpot, individual peptides eliciting activity demonstrated mean IFN-γ spot forming units per well (SFU)/1x10 5 cells of 115.5 (range 24.5-247.5). VST products were polyfunctional, releasing IFN-γ and TNF-α in response to identified epitopes, which were primarily HLA Class II restricted. Peptides from Human Parainfluenza Virus-1 corresponding to the HPIV3 epitopes showed cross-reactivity for HPIV1 in 11 of 12 tested epitopes (mean cross reactivity index: 1.19). Characterization of HPIV3 epitopes may enable development of third-party VSTs to treat immune suppressed patients with HPIV infection., (Copyright © 2020 Harris, Horn, Grant, Lang, Sani, Jensen-Wachspress, Kankate, Datar, Lazarski, Bollard and Keller.)

TCR-gamma delta (γδ) T-cells are considered important players in the graft-vs.-tumor effect following allogeneic hematopoietic cell transplantation (alloHCT) and have emerged as candidates for adoptive transfer immunotherapy in the treatment of both solid and hematological tumors. Systemic β-adrenergic receptor (β-AR) activation has been shown to mobilize TCR-γδ T-cells to the blood, potentially serving as an adjuvant for alloHCT and TCR-γδ T-cell therapy. We investigated if systemic β-AR activation, using acute dynamic exercise as an experimental model, can increase the mobilization, ex vivo expansion, and anti-tumor activity of TCR-γδ T-cells isolated from the blood of healthy humans. We also sought to investigate the β-AR subtypes involved, by administering a preferential β 1 -AR antagonist (bisoprolol) and a non-preferential β 1 + β 2 -AR antagonist (nadolol) prior to exercise as part of a randomized placebo controlled cross-over experiment. We found that exercise mobilized TCR-γδ cells to blood and augmented their ex vivo expansion by ~182% compared to resting blood when stimulated with IL-2 and ZOL for 14-days. Exercise also increased the proportion of CD56+, NKG2D+/CD62L-, CD158a/b/e+ and NKG2A- cells among the expanded TCR-γδ cells, and increased their cytotoxic activity against several tumor target cells (K562, U266, 221.AEH) in vitro by 40-60%. Blocking NKG2D on TCR-γδ cells in vitro eliminated the augmented cytotoxic effects of exercise against U266 target cells. Furthermore, administering a β 1 + β 2 -AR (nadolol), but not a β 1 -AR (bisoprolol) antagonist prior to exercise abrogated the exercise-induced enhancement in TCR-γδ T-cell mobilization and ex vivo expansion. Furthermore, nadolol completely abrogated while bisoprolol partially inhibited the exercise-induced increase in the cytotoxic activity of the expanded TCR-γδ T-cells. We conclude that acute systemic β-AR activation in healthy donors markedly augments the mobilization, ex vivo expansion, and anti-tumor activity of TCR-γδ T-cells and that some of these effects are due to β 2 -AR signaling and phenotypic shifts that promote a dominant activating signal via NKG2D. These findings highlight β-ARs as potential targets to favorably alter the composition of allogeneic peripheral blood stem cell grafts and improve the potency of TCR-γδ T-cell immune cell therapeutics., (Copyright © 2020 Baker, Bigley, Agha, Pedlar, O'Connor, Bond, Bollard, Katsanis and Simpson.)

Mycobacterial Infections can be severe in patients with T-cell deficiency or phagocyte disorders, and treatment is frequently complicated by antimicrobial resistance. Restoration of T-cell immunity via stem cell transplantation facilitates control of mycobacterial infections, but presence of active infections during transplantation is associated with a higher risk of mortality. Adoptive T cell immunotherapy has been successful in targeting viruses, but has not been attempted to treat mycobacterial infections. We sought to expand and characterize mycobacterial-specific T-cells derived from healthy donors in order to determine suitability for adoptive immunotherapy. Mycobacteria-specific T-cells (MSTs) were generated from 10 healthy donors using a rapid ex vivo expansion protocol targeting five known mycobacterial target proteins (AG85B, PPE68, ESXA, ESXB, and ADK). MSTs were compared to T-cells expanded from the same donors using lysate from M. tuberculosis or purified protein derivative from M. avium (sensitin). MST expansion from seven patients with primary immunodeficiency disorders (PID) and two patients with IFN-γ autoantibodies and invasive M. avium infections. MSTs expanded from healthy donors recognized a median of 3 of 5 antigens, with production of IFN-γ, TNF, and GM-CSF in CD4 + T cells. Comparison of donors who received BCG vaccine ( n = 6) to those who did not ( n = 4) showed differential responses to PPE68 ( p = 0.028) and ADK ( p = 0.015) by IFN-γ ELISpot. MSTs expanded from lysate or sensitin also recognized multiple mycobacterial antigens, with a statistically significant differences noted only in the response to PPE68 ( p = 0.016). MSTs expanded from patients with primary immunodeficiency (PID) and invasive mycobacterial infections showed activity against mycobacterial antigens in only two of seven subjects, whereas both patients with IFN-γ autoantibodies recognized mycobacterial antigens. Thus, MSTs can be generated from donors using a rapid expansion protocol regardless of history of BCG immunization. Most tested PID patients had no detectable T-cell immunity to mycobacteria despite history of infection. MSTs may have clinical utility for adoptive immunotherapy in T-cell deficient patients with invasive mycobacterial infections.

Patients with primary immunodeficiency disorders (PID) have an increased risk from acute and chronic Epstein-Barr Virus (EBV) viral infections and EBV-associated malignancies. Hematopoietic stem cell transplantation (HSCT) is a curative strategy for many patients with PID, but EBV-related complications are common in the immediate post-transplant period due to delayed reconstitution of T cell immunity. Adoptive T cell therapy with EBV-specific T cells is a promising therapeutic strategy for patients with PID both before and after HSCT. Here we review the methods used to manufacture EBV-specific T cells, the clinical outcomes, and the ongoing challenges for future development of the strategy.

While progress has been made in the treatment of both hematologic cancers and solid tumors, chemorefractory or relapsed disease often portends a dismal prognosis, and salvage chemotherapy or radiation expose patients to intolerable toxicities and may not be effective. Hematopoietic stem cell transplant offers the promise of cure for many patients, and while mismatched, unrelated or haploidentical donors are increasingly available, the recipients are at higher risk of severe immunosuppression and immune dysregulation due to graft versus host disease. Viral infections remain a primary cause of severe morbidity and mortality in this patient population. Again, many therapeutic options for viral disease are toxic, may be ineffective or generate resistance, or fail to convey long-term protection. Adoptive cell therapy with virus-specific T cells (VSTs) is a targeted therapy that is efficacious and has minimal toxicity in immunocompromised patients with CMV and EBV infections in particular. Products have since been generated specific for multiple viral antigens (multi-VST), which are not only effective but also confer protection in 70-90% of recipients when used as prophylaxis. Notably, these products can be generated from either virus-naive or virus-experienced autologous or allogeneic sources, including partially matched HLA-matched third-party donors. Obstacles to effective VST treatment are donor availability and product generation time. Banking of third-party VST is an attractive way to overcome these constraints and provide products on an as-needed basis. Other developments include epitope discovery to broaden the number of viral antigens targets in a single product, the optimization of VST generation from naive donor sources, and the modification of VSTs to enhance persistence and efficacy in vivo .