Natural Abs, which arise without known immune exposure, have been described that specifically recognize cells dying from apoptosis, but their role in innate immunity remains poorly understood. Herein, we show that the immune response to neoantigenic determinants on apoptotic thymocytes is dominated by Abs to oxidation-associated Ags, phosphorylcholine (PC), a head group that becomes exposed during programmed cell death, and malondialdehyde (MDA), a reactive aldehyde degradation product of polyunsaturated lipids produced following exposure to reactive oxidation species. While natural Abs to apoptotic cells in naive adult mice were dominated by PC and MDA specificities, the amounts of these Abs were substantially boosted by treatment of mice with apoptotic cells. Moreover, the relative amounts of PC and MDA Abs was affected by VH gene inheritance. Ab interactions with apoptotic cells also mediated the recruitment of C1q, which enhanced apoptotic cell phagocytosis by immature dendritic cells. Significantly, IgM Abs to both PC and MDA were primary factors in determining the efficiency of serum-dependent apoptotic cell phagocytosis. Hence, we demonstrate a mechanism by which certain natural Abs that recognize neoantigens on apoptotic cells, in naive mice and those induced by immune exposure to apoptotic cells, can enhance the functional capabilities of immature dendritic cells for phagocytic engulfment of apoptotic cells.

T cells expressing CD19-targeting chimeric antigen receptors (CARs) reveal high efficacy in the treatment of B cell malignancies. Here, we report that T cell receptor fusion constructs (TRuCs) comprising an antibody-based binding domain fused to T cell receptor (TCR) subunits can effectively reprogram an intact TCR complex to recognize tumor surface antigens. Unlike CARs, TRuCs become a functional component of the TCR complex. TRuC-T cells kill tumor cells as potently as second-generation CAR-T cells, but at significant lower cytokine release and despite the absence of an extra co-stimulatory domain. TRuC-T cells demonstrate potent anti-tumor activity in both liquid and solid tumor xenograft models. In several models, TRuC-T cells are more efficacious than respective CAR-T cells. TRuC-T cells are shown to engage the signaling capacity of the entire TCR complex in an HLA-independent manner.

SummaryHuman haematopoietic progenitor/stem cells (HPCs) differentiate into functional T cells in the thymus through a series of checkpoints. A convenient in vitro system will greatly facilitate the understanding of T-cell development and future engineering of therapeutic T cells. In this report, we established a lentiviral vector-engineered stromal cell line (LSC) expressing the key lymphopoiesis regulator Notch ligand, Delta-like 1 (DL1), as feeder cells (LSC-mDL1) supplemented with Flt3 ligand (fmslike tyrosine kinase 3, Flt3L or FL) and interleukin-7 for the development of T cells from CD34 + HPCs. We demonstrated T-cell development from human HPCs with various origins including fetal thymus (FT), fetal liver (FL), cord blood (CB) and adult bone marrow (BM). The CD34 + HPCs from FT, FL and adult BM expanded more than 100-fold before reaching the b-selection and CD4/CD8 double-positive T-cell stage. The CB HPCs, on the other hand, expanded more than 1000-fold before b-selection. Furthermore, the time required to reach b-selection differed for the various HPCs, 7 days for FT, 14 days for FL and CB, and 35 days for adult BM. Nevertheless, all of the T cells developed in vitro were stalled at the double-positive or immature single-positive stage with the exception that some CB-derived T cells arrived at a positive selection stage. Consequently, the LSC-mDL1 culture system illustrated diverse T-cell development potentials of pre-and post-natal and adult human BM HPCs. However, further modification of this in vitro T-cell development system is necessary to attain fully functional T cells.Keywords: bone marrow; cord blood; Delta-like 1; fetal tissue; lentiviral vector; lymphoid differentiation; T cellsPlease cite this article in press as: Patel E. et al. Diverse T-cell differentiation potentials of human fetal thymus, fetal liver, cord blood and adult bone marrow CD34 cells on lentiviral Delta-like-1-modified mouse stromal cells, Immunology (2009)

Interleukin (IL)-6 is a multi-functional cytokine that can either promote or suppress tissue inflammation depending on the specific disease context. IL-6 is elevated in the exocrine glands and serum of patients with Sjögren’s syndrome (SS), but the specific role of IL-6 in the pathogenesis of this disease has not been defined. In this study, we showed that IL-6 expression levels were increased with age in C56BL/6.NOD-Aec1Aec2 mice, a primary SS model, and higher than the control C57BL/6 mice. To assess the role of IL-6 during the immunological phase of SS development, a neutralizing anti-IL-6 antibody was administered into 16 week-old female C56BL/6.NOD-Aec1Aec2 mice, 3 times weekly for a consecutive 8 weeks. Neutralization of endogenous IL-6 throughout the immunological phase of SS development led to increased apoptosis, caspase-3 activation, leukocytic infiltration, and IFN-γ- and TNF-α production in the salivary gland. To further determine the effect of IL-6 on the apoptosis of exocrine gland cells, recombinant human IL-6 or the neutralizing anti-IL-6 antibody was injected into female C57BL/6 mice that received concurrent injection of anti-CD3 antibody to induce the apoptosis of exocrine glands. Neutralization of IL-6 enhanced, whereas administration of IL-6 inhibited apoptosis and caspase-3 activation in salivary and lacrimal glands in this model. The apoptosis-suppressing effect of IL-6 was associated with up-regulation of Bcl-xL and Mcl-1 in both glands. Moreover, IL-6 treatment induced activation of STAT3 and up-regulated Bcl-xL and Mcl-1 gene expression in a human salivary gland epithelial cell line. In conclusion, IL-6 inhibits the apoptosis of exocrine gland tissues and exerts a tissue-protective effect under inflammatory conditions including SS. These findings suggest the possibility of using this property of IL-6 to preserve exocrine gland tissue integrity and function under autoimmune and inflammatory conditions.

Dendritic cells (DCs) are important innate and adaptive immune effectors, and have a key role in antigen presentation and T-cell activation. Different lineages of DCs can be developed from hematopoietic progenitors following cytokine signaling, and the various lineages of DCs display distinct morphology, phenotype and functions. There has been limited information on differential cytokine-mediated molecular signaling in DCs. Analyses of surface molecules by flow cytometry and quantitative RNA profiling revealed differences between DCs derived from interleukin-4 (IL-4) versus IL-15 signaling, yet both lineages of DCs exhibited similar levels of surface molecules key to immune activation. Functional assays confirmed that IL-15-derived DCs elicited greater antigen-specific, primary and secondary CD8 and CD4 T-cell responses than did IL-4-derived DCs. Importantly, IL-15 DCs secreted substantial amounts of proinflammatory cytokines, including IL-6, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNFα), which helped polarize a strong T-cell response. Assessment of signaling pathways revealed that IL-15 DCs exhibited a lower levels of activated signal transducer and activator of transcription 5 (STAT5), STAT6 and extracellular signal-regulated kinase 1/2 than IL-4 DCs, but after lipopolysaccharide (LPS)/TNFα treatment, the STAT3 and p38 mitogen-activated protein kinase (MAPK) activities were significantly enhanced in the IL-15 DCs. Surprisingly, contrary to the canonical IL-15-mediated STAT5 signaling pathway in lymphoid cells, IL-15 did not mediate a strong STAT5 or STAT3 activation in DCs. Further analysis using specific inhibitors to STAT3 and p38 MAPK pathways revealed that the STAT3 signaling, but not p38 MAPK signaling, contributed to IFN-γ production in DCs. Therefore, while IL-15 does not promote the STAT signaling in DCs, the increased STAT3 activity after LPS/TNFα treatment of the IL-15 DCs has a key role in their high IFN-γ effector activities.