• Downregulation of SATB1 by miRNAs Reduces Megakaryocyte/Erythroid Progenitor Expansion in pre-clinical models of Diamond Blackfan Anemia Experimental Hematology Wilkes, M. C., Scanlon, V., Shibuya, A., Cepika, A., Eskin, A., Chen, Z., Narla, A., Glader, B., Roncarolo, M., Nelson, S. F., Sakamoto, K. M. 2022
  • Alloantigen-specific type 1 regulatory T cells suppress through CTLA-4 and PD-1 pathways and persist long-term in patients. Science translational medicine Chen, P. P., Cepika, A., Agarwal-Hashmi, R., Saini, G., Uyeda, M. J., Louis, D. M., Cieniewicz, B., Narula, M., Amaya Hernandez, L. C., Harre, N., Xu, L., Thomas, B. C., Ji, X., Shiraz, P., Tate, K. M., Margittai, D., Bhatia, N., Meyer, E., Bertaina, A., Davis, M. M., Bacchetta, R., Roncarolo, M. G. 2021; 13 (617): eabf5264


    [Figure: see text].

    View details for DOI 10.1126/scitranslmed.abf5264

    View details for PubMedID 34705520

  • Development of beta-globin gene correction in human hematopoietic stem cells as a potential durable treatment for sickle cell disease. Science translational medicine Lattanzi, A., Camarena, J., Lahiri, P., Segal, H., Srifa, W., Vakulskas, C. A., Frock, R. L., Kenrick, J., Lee, C., Talbott, N., Skowronski, J., Cromer, M. K., Charlesworth, C. T., Bak, R. O., Mantri, S., Bao, G., DiGiusto, D., Tisdale, J., Wright, J. F., Bhatia, N., Roncarolo, M. G., Dever, D. P., Porteus, M. H. 2021; 13 (598)


    Sickle cell disease (SCD) is the most common serious monogenic disease with 300,000 births annually worldwide. SCD is an autosomal recessive disease resulting from a single point mutation in codon six of the beta-globin gene (HBB). Ex vivo beta-globin gene correction in autologous patient-derived hematopoietic stem and progenitor cells (HSPCs) may potentially provide a curative treatment for SCD. We previously developed a CRISPR-Cas9 gene targeting strategy that uses high-fidelity Cas9 precomplexed with chemically modified guide RNAs to induce recombinant adeno-associated virus serotype 6 (rAAV6)-mediated HBB gene correction of the SCD-causing mutation in HSPCs. Here, we demonstrate the preclinical feasibility, efficacy, and toxicology of HBB gene correction in plerixafor-mobilized CD34+ cells from healthy and SCD patient donors (gcHBB-SCD). We achieved up to 60% HBB allelic correction in clinical-scale gcHBB-SCD manufacturing. After transplant into immunodeficient NSG mice, 20% gene correction was achieved with multilineage engraftment. The long-term safety, tumorigenicity, and toxicology study demonstrated no evidence of abnormal hematopoiesis, genotoxicity, or tumorigenicity from the engrafted gcHBB-SCD drug product. Together, these preclinical data support the safety, efficacy, and reproducibility of this gene correction strategy for initiation of a phase 1/2 clinical trial in patients with SCD.

    View details for DOI 10.1126/scitranslmed.abf2444

    View details for PubMedID 34135108

  • Hergen Spits-A legend at the top of his career. Allergy Mjosberg, J., Roncarolo, M. G., Blom, B. 2021

    View details for DOI 10.1111/all.14788

    View details for PubMedID 33751599

  • BHLHE40 Regulates IL-10 and IFN-γ Production in T Cells but Does Not Interfere With Human Type 1 Regulatory T Cell Differentiation Frontiers in Immunology Uyeda, M. J., Freeborn, R. A., Cieniewicz, B., Romano, R., Chen, P. P., Liu, J. M., Thomas, B., Lee, E., Cepika, A., Bacchetta, R., Roncarolo, M. 2021
  • Co-Expression of FOXP3FL and FOXP3Δ2 Isoforms Is Required for Optimal Treg-Like Cell Phenotypes and Suppressive Function. Frontiers in immunology Sato, Y., Liu, J., Lee, E., Perriman, R., Roncarolo, M. G., Bacchetta, R. 2021; 12: 752394


    FOXP3 is the master transcription factor in both murine and human FOXP3+ regulatory T cells (Tregs), a T-cell subset with a central role in controlling immune responses. Loss of the functional Foxp3 protein in scurfy mice leads to acute early-onset lethal lymphoproliferation. Similarly, pathogenic FOXP3 mutations in humans lead to immunodysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) syndrome, which are characterized by systemic autoimmunity that typically begins in the first year of life. However, although pathogenic FOXP3 mutations lead to overlapping phenotypic consequences in both systems, FOXP3 in human Tregs, but not mouse, is expressed as two predominant isoforms, the full length (FOXP3FL) and the alternatively spliced isoform, delta 2 (FOXP3Δ2). Here, using CRISPR/Cas9 to generate FOXP3 knockout CD4+ T cells (FOXP3KOGFP CD4+ T cells), we restore the expression of each isoform by lentiviral gene transfer to delineate their functional roles in human Tregs. When compared to FOXP3FL or FOXP3Δ2 alone, or double transduction of the same isoform, co-expression of FOXP3FL and FOXP3Δ2 induced the highest overall FOXP3 protein expression in FOXP3KOGFP CD4+ T cells. This condition, in turn, led to optimal acquisition of Treg-like cell phenotypes including downregulation of cytokines, such as IL-17, and increased suppressive function. Our data confirm that co-expression of FOXP3FL and FOXP3Δ2 leads to optimal Treg-like cell function and supports the need to maintain the expression of both when engineering therapeutics designed to restore FOXP3 function in otherwise deficient cells.

    View details for DOI 10.3389/fimmu.2021.752394

    View details for PubMedID 34737751

    View details for PubMedCentralID PMC8560788

  • The Yin and Yang of Type 1 Regulatory T Cells: From Discovery to Clinical Application. Frontiers in immunology Sayitoglu, E. C., Freeborn, R. A., Roncarolo, M. G. 2021; 12: 693105


    Regulatory T cells are essential players of peripheral tolerance and suppression of inflammatory immune responses. Type 1 regulatory T (Tr1) cells are FoxP3- regulatory T cells induced in the periphery under tolerogenic conditions. Tr1 cells are identified as LAG3+CD49b+ mature CD4+ T cells that promote peripheral tolerance through secretion of IL-10 and TGF-beta in addition to exerting perforin- and granzyme B-mediated cytotoxicity against myeloid cells. After the initial challenges of isolation were overcome by surface marker identification, ex vivo expansion of antigen-specific Tr1 cells in the presence of tolerogenic dendritic cells (DCs) and IL-10 paved the way for their use in clinical trials. With one Tr1-enriched cell therapy product already in a Phase I clinical trial in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT), Tr1 cell therapy demonstrates promising results so far in terms of efficacy and safety. In the current review, we identify developments in phenotypic and molecular characterization of Tr1 cells and discuss the potential of engineered Tr1-like cells for clinical applications of Tr1 cell therapies. More than 3 decades after their initial discovery, Tr1 cell therapy is now being used to prevent graft versus host disease (GvHD) in allo-HSCT and will be an alternative to immunosuppression to promote graft tolerance in solid organ transplantation in the near future.

    View details for DOI 10.3389/fimmu.2021.693105

    View details for PubMedID 34177953

  • Pre-clinical development and molecular characterization of an engineered type 1 regulatory T-cell product suitable for immunotherapy. Cytotherapy Liu, J. M., Chen, P., Uyeda, M. J., Cieniewicz, B., Sayitoglu, E. C., Thomas, B. C., Sato, Y., Bacchetta, R., Cepika, A. M., Roncarolo, M. G. 2021


    Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapeutic approach for many hematological disorders. However, allo-HSCT is frequently accompanied by a serious side effect: graft-versus-host disease (GVHD). The clinical use of allo-HSCT is limited by the inability of current immunosuppressive regimens to adequately control GvHD without impairing the graft-versus-leukemia effect (GvL) conferred by transplanted healthy immune cells. To address this, the authors have developed an engineered type 1 regulatory T-cell product called CD4IL-10 cells. CD4IL-10 cells are obtained through lentiviral transduction, which delivers the human IL10 gene into purified polyclonal CD4+ T cells. CD4IL-10 cells may provide an advantage over standard-of-care immunosuppressants because of the ability to suppress GvHD through continuous secretion of IL-10 and enhance the GvL effect in myeloid malignancies through targeted killing of malignant myeloid cells.Here the authors established a production process aimed at current Good Manufacturing Practice (cGMP) production for CD4IL-10 cells.The authors demonstrated that the CD4IL-10 cell product maintains the suppressive and cytotoxic functions of previously described CD4IL-10 cells. In addition, RNA sequencing analysis of CD4IL-10 identified novel transcriptome changes, indicating that CD4IL-10 cells primarily upregulate cytotoxicity-related genes. These include four molecules with described roles in CD8+ T and natural killer cell-mediated cytotoxicity: CD244, KLRD1, KLRC1 and FASLG. Finally, it was shown that CD4IL-10 cells upregulate IL-22, which mediates wound healing and tissue repair, particularly in the gut.Collectively, these results pave the way toward clinical translation of the cGMP-optimized CD4IL-10 cell product and uncover new molecules that have a role in the clinical application of CD4IL-10 cells.

    View details for DOI 10.1016/j.jcyt.2021.05.010

    View details for PubMedID 34404616

  • InsB9-23 Gene Transfer To Hepatocytes-Based Combined Therapy Abrogates Recurrence of Type-1 Diabetes After Islet Transplantation. Diabetes Russo, F., Citro, A., Squeri, G., Sanvito, F., Monti, P., Gregori, S., Roncarolo, M. G., Annoni, A. 2020


    The induction of antigen (Ag)-specific tolerance represents a therapeutic option for autoimmune diabetes. We demonstrated that administration of lentiviral vector enabling expression of insulinB9-23 (LV.InsB) in hepatocytes, arrests beta cell destruction in pre-diabetic NOD mice, by generating InsB9-23-specific FoxP3+T regulatory cells (Tregs). LV.InsB in combination with a suboptimal dose of anti-CD3 mAb (combined therapy, 1X5g CT5) reverts diabetes and prevents recurrence of autoimmunity following islets transplantation in 50% of NOD mice. We investigated whether CT optimization could lead to abrogation of recurrence of autoimmunity. Therefore, allo-islets were transplanted after optimized CT tolerogenic conditioning (1X25g CT25). Diabetic NOD mice conditioned with CT25 when glycaemia was <500mg/dL, remained normoglycaemic for 100 days after allo-islets transplantation, displayed reduced insulitis, but independently from the graft. Accordingly, cured mice showed T cell unresponsiveness to InsB9-23 stimulation and increased Tregs frequency in islets infiltration and pancreatic LN. Additional studies revealed a complex mechanism of Ag-specific immune regulation driven by CT25, in which both Tregs and PDL1 co-stimulation cooperate to control diabetogenic cells, while transplanted islets play a crucial role, although transient, recruiting diabetogenic cells. Therefore, CT25 before allo-islets transplantation represents an Ag-specific immunotherapy to resolve autoimmune diabetes in the presence of residual endogenous beta cell mass.

    View details for DOI 10.2337/db19-1249

    View details for PubMedID 33122392

  • Celebrating 20 years of FOCIS. Science immunology Roncarolo, M. G., Anderson, M. S. 2020; 5 (52)

    View details for DOI 10.1126/sciimmunol.abe8102

    View details for PubMedID 33037068

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