Thymic regulatory T cells arise via two distinct developmental programs.

The developmental programs that generate a broad repertoire of regulatory T cells (Treg cells) able to respond to both self antigens and non-self antigens remain unclear. Here we found that mature Treg cells were generated through two distinct developmental programs involving CD25+ Treg cell progenitors (CD25+ TregP cells) and Foxp3lo Treg cell progenitors (Foxp3lo TregP cells). CD25+ TregP cells showed higher rates of apoptosis and interacted with thymic self antigens with higher affinity than did Foxp3lo TregP cells, and had a T cell antigen receptor repertoire and transcriptome distinct from that of Foxp3lo TregP cells. The development of both CD25+ TregP cells and Foxp3lo TregP cells was controlled by distinct signaling pathways and enhancers. Transcriptomics and histocytometric data suggested that CD25+ TregP cells and Foxp3lo TregP cells arose by coopting negative-selection programs and positive-selection programs, respectively. Treg cells derived from CD25+ TregP cells, but not those derived from Foxp3lo TregP cells, prevented experimental autoimmune encephalitis. Our findings indicate that Treg cells arise through two distinct developmental programs that are both required for a comprehensive Treg cell repertoire capable of establishing immunotolerance.

Molecular and Functional Characterization of Lymphoid Progenitor Subsets Reveals a Bipartite Architecture of Human Lymphopoiesis.

The classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127- and CD127+ early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127- and CD127+ ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127- ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127+ ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis.

IL-15 superagonist N-803 improves IFNγ production and killing of leukemia and ovarian cancer cells by CD34+ progenitor-derived NK cells.

Allogeneic natural killer (NK) cell transfer is a potential immunotherapy to eliminate and control cancer. A promising source are CD34 + hematopoietic progenitor cells (HPCs), since large numbers of cytotoxic NK cells can be generated. Effective boosting of NK cell function can be achieved by interleukin (IL)-15. However, its in vivo half-life is short and potent trans-presentation by IL-15 receptor α (IL-15Rα) is absent. Therefore, ImmunityBio developed IL-15 superagonist N-803, which combines IL-15 with an activating mutation, an IL-15Rα sushi domain for trans-presentation, and IgG1-Fc for increased half-life. Here, we investigated whether and how N-803 improves HPC-NK cell functionality in leukemia and ovarian cancer (OC) models in vitro and in vivo in OC-bearing immunodeficient mice. We used flow cytometry-based assays, enzyme-linked immunosorbent assay, microscopy-based serial killing assays, and bioluminescence imaging, for in vitro and in vivo experiments. N-803 increased HPC-NK cell proliferation and interferon (IFN)γ production. On leukemia cells, co-culture with HPC-NK cells and N-803 increased ICAM-1 expression. Furthermore, N-803 improved HPC-NK cell-mediated (serial) leukemia killing. Treating OC spheroids with HPC-NK cells and N-803 increased IFNγ-induced CXCL10 secretion, and target killing after prolonged exposure. In immunodeficient mice bearing human OC, N-803 supported HPC-NK cell persistence in combination with total human immunoglobulins to prevent Fc-mediated HPC-NK cell depletion. Moreover, this combination treatment decreased tumor growth. In conclusion,  N-803 is a promising IL-15-based compound that boosts HPC-NK cell expansion and functionality in vitro and in vivo. Adding N-803 to HPC-NK cell therapy could improve cancer immunotherapy.

Intrathymic transplantation of bone marrow-derived progenitors provides long-term thymopoiesis.

The sustained differentiation of T cells in the thymus cannot be maintained by resident intrathymic (IT) precursors and requires that progenitors be replenished from the bone marrow (BM). In patients with severe combined immunodeficiency (SCID) treated by hematopoietic stem cell transplantation, late T-cell differentiation defects are thought to be due to an insufficient entry of donor BM progenitors into the thymus. Indeed, we find that the intravenous injection of BM progenitors into nonconditioned zeta-chain-associated protein kinase 70 (ZAP-70)-deficient mice with SCID supports short- but not long-term thymopoiesis. Remarkably, we now show that the IT administration of these progenitors produces a significant level of donor-derived thymopoiesis for more than 6 months after transplantation. In contrast to physiologic thymopoiesis, long-term donor thymopoiesis was not due to the continued recruitment of progenitors from the BM. Rather, IT transplantation resulted in the unique generation of a large population of early c-Kit(high) donor precursors within the thymus. These ZAP-70-deficient mice that received an IT transplant had a significantly increased prothymocyte niche compared with their untreated counterparts; this phenotype was associated with the generation of a medulla. Thus, IT administration of BM progenitors results in the filling of an expanded precursor niche and may represent a strategy for enhancing T-cell differentiation in patients with SCID.

Polyclonal T-cell reconstitution of X-SCID recipients after in utero transplantation of lymphoid-primed multipotent progenitors.

Although successful in utero hematopoietic cell transplantation (IUHCT) of X-linked severe combined immune deficiency (X-SCID) with enriched stem and progenitor cells was achieved more than a decade ago, it remains applied only in rare cases. Although this in part reflects that postnatal transplantations have overall given good results, there are no direct comparisons between IUHCT and postnatal transplantations of X-SCID. The proposed tolerance of the fetal immune system to foreign human leukocyte antigen early in gestation, a main rationale behind IUHCT, has recently been challenged by evidence for a considerable immune barrier against in utero transplanted allogeneic bone marrow cells. Consequently, there is need for further exploring the application of purified stem and progenitor cells to overcome this barrier also in IUHCT. Herein, we demonstrate in a congenic setting that recently identified lymphoid-primed multipotent progenitors are superior to hematopoietic stem cells in providing rapid lymphoid reconstitution after IUHCT of X-SCID recipients, and sustain in the long-term B cells, polyclonal T cells, as well as short-lived B-cell progenitors and thymic T-cell precursors. We further provide evidence for IUHCT of hematopoietic stem cells giving superior B- and T-cell reconstitution in fetal X-SCID recipients compared with neonatal and adolescent recipients.

Determination of the Fate and Function of Innate Lymphoid Cells Following Adoptive Transfer of Innate Lymphoid Cell Precursors.

Innate lymphoid cells are a heterogeneous family of tissue-resident and circulating lymphocytes that play an important role in host immunity. Recent studies have profiled the developmental pathways of mature ILCs and have identified ILC progenitors in the bone marrow through the use of transcription factor reporter mice. Here we describe methodology to identify and isolate bone marrow CHILP and ILC2 progenitor (ILC2P) cells based on cell surface marker expression for adoptive transfer into lymphopenic mice to track the fate of developing ILCs.

Mitochondrial Membrane Potential Identifies Cells with Enhanced Stemness for Cellular Therapy.

Long-term survival and antitumor immunity of adoptively transferred CD8(+) T cells is dependent on their metabolic fitness, but approaches to isolate therapeutic T cells based on metabolic features are not well established. Here we utilized a lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM) to identify and isolate metabolically robust T cells based on their mitochondrial membrane potential (ΔΨm). Comprehensive metabolomic and gene expression profiling demonstrated global features of improved metabolic fitness in low-ΔΨm-sorted CD8(+) T cells. Transfer of these low-ΔΨm T cells was associated with superior long-term in vivo persistence and an enhanced capacity to eradicate established tumors compared with high-ΔΨm cells. Use of ΔΨm-based sorting to enrich for cells with superior metabolic features was observed in CD8(+), CD4(+) T cell subsets, and long-term hematopoietic stem cells. This metabolism-based approach to cell selection may be broadly applicable to therapies involving the transfer of HSC or lymphocytes for the treatment of viral-associated illnesses and cancer.

New cell sources for T cell engineering and adoptive immunotherapy.

The promising clinical results obtained with engineered T cells, including chimeric antigen receptor (CAR) therapy, call for further advancements to facilitate and broaden their applicability. One potentially beneficial innovation is to exploit new T cell sources that reduce the need for autologous cell manufacturing and enable cell transfer across histocompatibility barriers. Here we review emerging T cell engineering approaches that utilize alternative T cell sources, which include virus-specific or T cell receptor-less allogeneic T cells, expanded lymphoid progenitors, and induced pluripotent stem cell (iPSC)-derived T lymphocytes. The latter offer the prospect for true off-the-shelf, genetically enhanced, histocompatible cell therapy products.

Generation, isolation, and engraftment of in vitro-derived human T cell progenitors.

T cells typically differentiate via a series of coordinated steps within the highly specialized microenvironment of the thymus. Traditionally, human T-lymphopoiesis in vitro has been studied using the hybrid human/mouse fetal thymic organ culture system. Pioneering work by McCune et al. devised a method to examine human T cell development in vivo in relation to HIV-1 using the SCID/hu (thy/liv) model. This was followed by models that better reflected the ability of human hematopoietic cells to home and differentiate within the mouse host without human fetal tissues; however, human T cell development in these animals was poor. In this chapter, we outline a procedure to generate human progenitor T (proT) cells in vitro from umbilical cord blood-derived hematopoietic stem cells using the OP9-DL1 cell system; in addition, we describe the method used to examine the engraftment of in vitro-derived proT cells into immunodeficient mouse strains.

New insights into the pathogenesis of adenosine deaminase-severe combined immunodeficiency and progress in gene therapy.

PURPOSE OF REVIEW: Adenosine deaminase (ADA)- deficient severe combined immunodeficiency (SCID) is a complex metabolic and immunological disorder, characterized by a severe immunodeficiency due to the accumulation of purine metabolites in plasma and cells. This review summarizes recent findings on the pathogenesis of immunological and nonimmunological defects in ADA deficiency and the successful outcome of gene therapy trials for this condition. RECENT FINDINGS: Recent reports show that ADA-SCID is associated with an increased frequency of autoimmune manifestations and high risk of central nervous system (CNS) complications even after bone marrow transplantation. It remains unclear to what extent infection-related or disease-specific factors correlate with this divergent outcome.Recent trials represented the first demonstration of long-term clinical efficacy of HSC gene therapy for ADA-SCID, underlining that gene therapy has a favorable safety profile and is effective in restoring normal purine metabolism and immune functions. Molecular studies showed that the retroviral integration profile after successful gene therapy did not cause selection or expansion of malignant cell clones in vivo. SUMMARY: Gene therapy for ADA-deficient SCID is an effective treatment, providing long-term clinical benefit for affected patients. Future research will be needed to address the occurrence of autoimmune manifestations and nonimmunological defects in order to improve patients' long-term prospects.