Interferon priming is essential for human CD34+ cell-derived plasmacytoid dendritic cell maturation and function.

Plasmacytoid dendritic cells (pDC) are essential for immune competence. Here we show that pDC precursor differentiated from human CD34+ hematopoietic stem and progenitor cells (HSPC) has low surface expression of pDC markers, and has limited induction of type I interferon (IFN) and IL-6 upon TLR7 and TLR9 agonists treatment; by contrast, cGAS or RIG-I agonists-mediated activation is not altered. Importantly, after priming with type I and II IFN, these precursor pDCs attain a phenotype and functional activity similar to that of peripheral blood-derived pDCs. Data from CRISPR/Cas9-mediated genome editing of HSPCs further show that HSPC-pDCs with genetic modifications can be obtained, and that expression of the IFN-α receptor is essential for the optimal function, but dispensable for the differentiation, of HSPC-pDC percursor. Our results thus demonstrate the biological effects of IFNs for regulating pDC function, and provide the means of generating of gene-modified human pDCs.

The TLR9 agonist MGN1703 triggers a potent type I interferon response in the sigmoid colon.

Toll-like receptor 9 (TLR9) agonists are being developed for treatment of colorectal and other cancers, yet the impact of these drugs on human intestines remains unknown. This, together with the fact that there are additional potential indications for TLR9 agonist therapy (e.g., autoimmune and infectious diseases), led us to investigate the impact of MGN1703 (Lefitolimod) on intestinal homeostasis and viral persistence in HIV-positive individuals. Colonic sigmoid biopsies were collected (baseline and week four) from 11 HIV+ individuals on suppressive antiretroviral therapy, who received MGN1703 (60 mg s.c.) twice weekly for 4 weeks in a single-arm, phase 1b/2a study. Within sigmoid mucosa, global transcriptomic analyses revealed 248 modulated genes (false discovery rate<0.05) including many type I interferon (IFN)-stimulated genes. MGN1703 increased the frequencies of cells exhibiting MX1 (P=0.001) and ISG15 (P=0.014) protein expression. No changes were observed in neutrophil infiltration (myeloperoxidase; P=0.97). No systematic effect on fecal microbiota structure was observed (analysis of similarity Global R=-0.105; P=0.929). TLR9 expression at baseline was inversely proportional to the change in integrated HIV DNA during MGN1703 treatment (P=0.020). In conclusion, MGN1703 induced a potent type I IFN response, without a concomitant general inflammatory response, in the intestines.

IL-2 receptor γ-chain molecule is critical for intestinal T-cell reconstitution in humanized mice.

Intestinal immune cells are important in host defense, yet the determinants for human lymphoid homeostasis in the intestines are poorly understood. In contrast, lymphoid homeostasis has been studied extensively in mice, where the requirement for a functional common γ-chain molecule has been established. We hypothesized that humanized mice could offer insights into human intestinal lymphoid homeostasis if generated in a strain with an intact mouse common γ-chain molecule. To address this hypothesis, we used three mouse strains (non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) (N/S); NOD/SCID γ-chain(-/-) (NSG); and Rag2(-/-) γ-chain(-/-) (DKO)) and two humanization techniques (bone marrow liver thymus (BLT) and human CD34(+) cell bone marrow transplant of newborn mice (hu)) to generate four common types of humanized mice: N/S-BLT, NSG-BLT, NSG-hu, and DKO-hu mice. The highest levels of intestinal human T cells throughout the small and large intestines were observed in N/S-BLT mice, which have an intact common γ-chain molecule. Furthermore, the small intestine lamina propria T-cell populations of N/S-BLT mice exhibit a human intestine-specific surface phenotype. Thus, the extensive intestinal immune reconstitution of N/S-BLT mice was both quantitatively and qualitatively better when compared with the other models tested such that N/S-BLT mice are well suited for the analysis of human intestinal lymphocyte trafficking and human-specific diseases affecting the intestines.

Functional and phenotypic characterization of the humanized BLT mouse model.

T cells play a central role in the development of immune responses. Patients lacking T cells because of genetic defects such as DiGeorge or Nezelof syndromes and patients infected with the human immunodeficiency virus are highly susceptible to infections and cancers. The lack of adequate in vivo models of T cell neogenesis have hindered the development and clinical implementation of effective therapeutic modalities aimed at treating these and other clinically important maladies. Transplantation of severe combined immunodeficient (SCID) mice with human hematopoietic stem cells results in long-term engraftment and systemic reconstitution with human progenitor, B, and myeloid cells, but curiously, human T cells are rarely present in any tissue. While the implantation of SCID mice with human fetal thymus and liver (SCID-hu thy/liv mice) allows the development of abundant thymocytes that are localized in the human organoid implant, there is minimal systemic repopulation with human T cells. However, we have recently shown that transplantation of autologous human hematopoietic fetal liver CD34+ cells into the nonobese diabetic (NOD)/SCID mouse background previously implanted with fetal thymic and liver tissues results in long-term, systemic human T cell homeostasis. In addition to human T cells, these mice have systemic repopulation with human B cells, monocytes/macrophages, and dendritic cells (DC). Importantly, in these mice the T cells developed in the human thymic implant are capable of being activated by human antigen-presenting cells and mount potent human MHC-restricted T cell immune responses.

Susceptibility to Theiler's murine encephalomyelitis virus-induced demyelinating disease in BALB/cAnNCr mice is related to absence of a CD4+ T-cell subset.

Two histocompatible substrains of BALB/c mice (BALB/cByJ, BALB/cAnNCr) are resistant and susceptible, respectively, to Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-1DD)--a model for viral etiology of human multiple sclerosis. BALB/cByJ mice become susceptible following low-dose irradiation given prior to infection. Resistance is restored by adoptive transfer of CD8+ (but not CD4+) splenic T cells from infected, unirradiated BALB/cByJ donors. In contrast resistance is conferred to BALB/cAnNCr mice by adoptive transfer of either CD4+ or CD8+ T cells from resistant BALB/cByJ donors. T cells from BALB/cAnNCr mice cannot confer protection. To integrate these two observations, we hypothesized that the BALB/cAnNCr mice possess precursors of the regulatory CD8+ T cells, but fail to activate them because they lack a critical CD4+ T-cell subpopulation. We tested this model using serial transfers. The transfer of CD4+ T cells from the BALB/cByJ to the BALB/cAnNCr mice permitted development of BALB/cAnNCr CD8+ T cells that in turn, provided resistance when transferred into susceptible recipients. The BALB/cByJ CD4+ T cells, which activated the CD8+ cells, were sensitive to low-dose irradiation, unlike CD4+ T cells involved in the later inflammatory demyelination. Thus, susceptibility of BALB/cAnNCr mice is due to a defective/absent CD4+ T-cell subset acting immediately after infection.