Search

Introduction: Refractory/relapsed pediatric acute leukemia are still clinically challenging and new therapeutic strategies are needed. Interactions between Natural Killer Group 2D (NKG2D) receptor, expressed in cytotoxic immune cells, and its ligands (NKG2DL), which are upregulated in leukemic blasts, are important for anti-leukemia immunosurveillance. Nevertheless, leukemia cells may develop immunoescape strategies as NKG2DL shedding and/or downregulation., Methods: In this report, we analyzed the anti-leukemia activity of NKG2D chimeric antigen receptor (CAR) redirected memory (CD45RA - ) T cells in vitro and in a murine model of T-cell acute lymphoblastic leukemia (T-ALL). We also explored in vitro how soluble NKG2DL (sNKG2DL) affected NKG2D-CAR T cells' cytotoxicity and the impact of NKG2D-CAR T cells on Jurkat cells gene expression and in vivo functionality., Results: In vitro , we found NKG2D-CAR T cells targeted leukemia cells and showed resistance to the immunosuppressive effects exerted by sNKG2DL. In vivo , NKG2D-CAR T cells controlled T cell leukemia burden and increased survival of the treated mice but failed to cure the animals. After CAR T cell treatment, Jurkat cells upregulated genes related to proliferation, survival and stemness, and in vivo , they exhibited functional properties of leukemia initiating cells., Discussion: The data here presented suggest, that, in combination with other therapeutic approaches, NKG2D-CAR T cells could be a novel treatment for pediatric T-ALL., Competing Interests: MB-P was previously employed by the company Medpace Germany, GmbH, though not at the time of the study, and is currently employed by the company Medpace Spain. 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. The handling editor FM declared a shared committee the National Network of Advanced therapies TerAv with the author AP-M at the time of review., (Copyright © 2023 Ibáñez-Navarro, Fernández, Escudero, Esteso, Campos-Silva, Navarro-Aguadero, Leivas, Caracuel, Rodríguez-Antolín, Ortiz, Navarro-Zapata, Mestre-Durán, Izquierdo, Balaguer-Pérez, Ferreras, Martínez-López, Valés-Gómez, Pérez-Martínez and Fernández.)

Background: Inborn errors of immunity (IEI) are a group of monogenic diseases that confer susceptibility to infection, autoimmunity, and cancer. Despite the life-threatening consequences of some IEI, their genetic cause remains unknown in many patients., Objective: We investigated a patient with an IEI of unknown genetic etiology., Methods: Whole-exome sequencing identified a homozygous missense mutation of the gene encoding ezrin (EZR), substituting a threonine for an alanine at position 129., Results: Ezrin is one of the subunits of the ezrin, radixin, and moesin (ERM) complex. The ERM complex links the plasma membrane to the cytoskeleton and is crucial for the assembly of an efficient immune response. The A129T mutation abolishes basal phosphorylation and decreases calcium signaling, leading to complete loss of function. Consistent with the pleiotropic function of ezrin in myriad immune cells, multidimensional immunophenotyping by mass and flow cytometry revealed that in addition to hypogammaglobulinemia, the patient had low frequencies of switched memory B cells, CD4 + and CD8 + T cells, MAIT, γδ T cells, and central naive CD4 + cells., Conclusions: Autosomal-recessive human ezrin deficiency is a newly recognized genetic cause of B-cell deficiency affecting cellular and humoral immunity., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background Aims: We have previously demonstrated the safety and feasibility of adoptive cell therapy with CD45RA - memory T cells containing severe acute respiratory syndrome coronavirus 2-specific T cells for patients with coronavirus disease 2019 from an unvaccinated donor who was chosen based on human leukocyte antigen compatibility and cellular response. In this study, we examined the durability of cellular and humoral immunity within CD45RA - memory T cells and the effect of dexamethasone, the current standard of care treatment, and interleukin-15, a cytokine critically involved in T-cell maintenance and survival., Methods: We performed a longitudinal analysis from previously severe acute respiratory syndrome coronavirus 2-infected and infection-naïve individuals covering 21 months from infection and 10 months after full vaccination with the BNT162b2 Pfizer/BioNTech vaccine., Results: We observed that cellular responses are maintained over time. Humoral responses increased after vaccination but were gradually lost. In addition, dexamethasone did not alter cell functionality or proliferation of CD45RA -  T cells, and interleukin-15 increased the memory T-cell activation state, regulatory T cell expression, and interferon gamma release., Conclusions: Our results suggest that the best donors for adoptive cell therapy would be recovered individuals and 2 months after vaccination, although further studies with larger cohorts would be needed to confirm this finding. Dexamethasone did not affect the characteristics of the memory T cells at a concentration used in the clinical practice and IL-15 showed a positive effect on SARS-CoV-2-specific CD45RA -  T cells., Competing Interests: Declaration of Competing Interest AP-M and CF filed patent EP20382850 on Memory T cells as adoptive cell therapy for viral diseases. All other authors declare no competing interest., (Copyright © 2022 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Introduction: Natural killer (NK) cells are lymphocytes from the innate immune system part of the first defense barrier against infected and transformed cells, representing 5%-15% of peripheral blood lymphocytes. The cytotoxic capacity of NK cells is controlled by a balance between inhibitory and activating NK receptors expressed on their surface, which recognize and interact with the ligands on stressed cells. The cytokines involved in NK cell activation, proliferation, survival, and cytotoxicity are signaled mainly through the Janus kinase and signal transducer and activator of transcription proteins (JAK/STAT) pathway. NK cells are also activated in response to pathogens through Toll-like receptors (TLRs) expressed on their surface. Ruxolitinib is a specific JAK1/2 inhibitor approved for treating myelofibrosis and for steroid-refractory acute and chronic graft-versus-host disease (SR-GvHD)., Methods: Purified NK cells from healthy donors were stimulated with two TOLL-like receptor ligands, LPS and CpG, in the presence of different concentrations of Ruxolitinib., Results: This study showed the effects of ruxolitinib on TLR4 and TLR9 ligand-activated NK cells from healthy donors. Ruxolitinib did not completely inhibit STAT3 phosphorylation and had a moderate effect on NK cell cytokine activation via the TLR pathway. Only the highest doses of ruxolitinib led to a decrease in the pro-inflammatory cytokines tumor necrosis factor α, interferon-γ, interleukin-6, and interleukin-1β. The cytotoxic capacity of stimulated NK cells versus K562, SEM, and MV-4-11 cell lines was reduced by increasing doses of ruxolitinib, but it was not completely abolished and we observed no major changes in degranulation capacity. Phenotypic changes were observed in activated NK cells in the presence of ruxolitinib. In a small cohort of pediatric patients treated with ruxolitinib for SR-GvHD, we observed no decrease in NK cell counts; however, further prospective studies with larger cohorts are necessary to confirm this finding., Discussion: In summary, our results showed that the functional capabilities and phenotype of NK cells activated through TLR4/9 agonists were not completely abolished by the inhibition of the JAK-STAT pathway by ruxolitinib., Competing Interests: The 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 Mestre-Durán, Martín-Cortázar, García-Solís, Pernas, Pertíñez, Galán, Sisinni, Clares-Villa, Navarro-Zapata, Al-Akioui, Escudero, Ferreras and Pérez-Martínez.)

Chimeric Antigen Receptor (CAR) T-cell therapy has demonstrated high rates of response in recurrent B-cell Acute Lymphoblastic Leukemia in children and young adults. Despite this success, a fraction of patients' experience relapse after treatment. Relapse is often preceded by recovery of healthy B cells, which suggests loss or dysfunction of CAR T-cells in bone marrow. This site is harder to access, and thus is not monitored as frequently as peripheral blood. Understanding the interplay between B cells, leukemic cells, and CAR T-cells in bone marrow is paramount in ascertaining the causes of lack of response. In this paper, we put forward a mathematical model representing the interaction between constantly renewing B cells, CAR T-cells, and leukemic cells in the bone marrow. Our model accounts for the maturation dynamics of B cells and incorporates effector and memory CAR T-cells. The model provides a plausible description of the dynamics of the various cellular compartments in bone marrow after CAR T infusion. After exploration of the parameter space, we found that the dynamics of CAR T product and disease were independent of the dose injected, initial B-cell load, and leukemia burden. We also show theoretically the importance of CAR T product attributes in determining therapy outcome, and have studied a variety of possible response scenarios, including second dosage schemes. We conclude by setting out ideas for the refinement of the model.

Syndrome coronavirus 2 (SARS-CoV-2) pandemic is causing a second outbreak significantly delaying the hope for the virus' complete eradication. In the absence of effective vaccines, we need effective treatments with low adverse effects that can treat hospitalized patients with COVID-19 disease. In this study, we determined the existence of SARS-CoV-2-specific T cells within CD45RA - memory T cells in the blood of convalescent donors. Memory T cells can respond quickly to infection and provide long-term immune protection to reduce the severity of COVID-19 symptoms. Also, CD45RA - memory T cells confer protection from other pathogens encountered by the donors throughout their life. It is of vital importance to resolve other secondary infections that usually develop in patients hospitalized with COVID-19. We found SARS-CoV-2-specific memory T cells in all of the CD45RA - subsets (CD3 + , CD4 + , and CD8 + ) and in the central memory and effector memory subpopulations. The procedure for obtaining these cells is feasible, easy to implement for small-scale manufacture, quick and cost-effective, involves minimal manipulation, and has no GMP requirements. This biobank of specific SARS-CoV-2 memory T cells would be immediately available "off-the-shelf" to treat moderate/severe cases of COVID-19, thereby increasing the therapeutic options available for these patients., Competing Interests: CF, BS, and AP-M filed a patent on this topic. BS received fees from Celgene, Gilead, Sanofi and Novo-Nordisk unrelated to this work. 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 © 2021 Ferreras, Pascual-Miguel, Mestre-Durán, Navarro-Zapata, Clares-Villa, Martín-Cortázar, De Paz, Marcos, Vicario, Balas, García-Sánchez, Eguizabal, Solano, Mora-Rillo, Soria and Pérez-Martínez.)

Natural killer (NK) cells represent promising tools for cancer immunotherapy. We report the optimization of an NK cell activation-expansion process and its validation on clinical-scale., Methods: RPMI-1640, stem cell growth medium (SCGM), NK MACS and TexMACS were used as culture mediums. Activated and expanded NK cells (NKAE) were obtained by coculturing total peripheral blood mononuclear cells (PBMC) or CD45RA + cells with irradiated K562mbIL15-41BBL or K562mbIL21-41BBL. Fold increase, NK cell purity, activation status, cytotoxicity and transcriptome profile were analyzed. Clinical-grade NKAE cells were manufactured in CliniMACS Prodigy., Results: NK MACS and TexMACs achieved the highest NK cell purity and lowest T cell contamination. Obtaining NKAE cells from CD45RA + cells was feasible although PBMC yielded higher total cell numbers and NK cell purity than CD45RA + cells. The highest fold expansion and NK purity were achieved by using PBMC and K562mbIL21-41BBL cells. However, no differences in activation and cytotoxicity were found when using either NK cell source or activating cell line. Transcriptome profile showed to be different between basal NK cells and NKAE cells expanded with K562mbIL21-41BBL or K562mbIL15-41BBL. Clinical-grade manufactured NKAE cells complied with the specifications from the Spanish Regulatory Agency., Conclusions: GMP-grade NK cells for clinical use can be obtained by using different starting cells and aAPC.

Intestinal passenger T leukocytes are responsible for graft-versus-host disease (GvHD) in intestinal transplantation (ITx). We hypothesized that ex vivo fludarabine treatment of the bowel graft would diminish the risk of GvHD and improve overall survival post-transplant. We performed isolated heterotopic small bowel transplantations from Lewis (LEW) to Brown Norway (BN) rat strains, which generated GvHD signs from the fourth day post-transplant. These symptoms included rash, weight loss, piloerection, and diarrhea. The grafts of one of the experimental groups were immersed and sealed in cold Celsior preservation solution with 1000 µm fludarabine for 1 h, prior to its implantation into recipient animals. No histological signs of intestinal tissue alterations were observed after fludarabine treatment. Fludarabine-treated bowel recipients showed significantly later and milder clinical signs of GvHD and reduced total donor cell chimerism, as determined by flow cytometry using strain-specific anti-HLA antibodies. Additionally, fludarabine treatment prolonged recipients' overall survival (13.5 days ± 0.3 days vs. 9.2 days ± 0.5). We conclude that active modification of the intestinal leukocyte composition is advantageous in our ITx animal model. Immunosuppression with fludarabine during the surgical procedure, which could be translated directly to the clinic, protects bowel recipients from GvHD and improves overall post-transplant survival., (© 2020 Steunstichting ESOT. Published by John Wiley & Sons Ltd.)