Stained cells were washed with Perm/Wash and subsequently with PBS before measurement of fluorescent intensity on an LSR Fortessa (BD Biosciences; Frederick, MD, USA). cells expressed more TLR9 than adult na?ve B cells. Neonatal na?ve B cells demonstrated impaired TLR2- and TLR7- but enhanced TLR9-mediated cytokine production. Significantly fewer newborn na?ve B cells underwent CSR to produce IgG, an impairment also noted with IL-21 stimulation. Additionally, co-stimulation via CD40 and TLRs induced greater cytokine production in adult B cells. Thus, while newborn na?ve B cells demonstrate adult-level expression of TLRs and CD40, the responses to stimulation of these receptors are distinct. Relatively high expression of TLR9 and impaired CD40-mediated Ig secretion contributes to distinct innate and adaptive immunity of human newborns and may inform novel approaches to early life immunization. Keywords: B lymphocyte, TLR, cytokine, neonatal, class-switch recombination Introduction B cells play a critical role in protection from infectious diseases. While Cav1.3 they are best known for their contribution to adaptive immunity via production of soluble immunoglobulins (antibodies, Igs or Abs), innate responses of B cells also impact the subsequent adaptive immune response.1 B cells SMIP004 express TLRs,2 a family (10 members in humans) of innate pattern-recognition receptors (PRRs) that recognize conserved microbial molecular patterns and initiate MyD88-dependent and -independent signaling events,3 and stimulation of B cell TLRs can induce cytokine production and influence their proliferation and activation.4 In particular, B cells express TLR9, which is responsive to CpG motif-rich bacterial DNA and to synthetic CpG DNA.5 While maternal Abs provide some protection from infection at birth, endogenous neonatal B cell responses to immunization and infection are often inadequate and as circulating levels of maternal IgG diminish with age (half-life ~21C30 days) this contributes to a general susceptibility to infection.6 Overall, innate immune responses of murine and human neonatal leukocytes such as monocytes and dendritic cells are different from adult counterparts,7, 8 with characteristic impaired inflammatory/Th1-polarizing cytokine (e.g., TNF and IL-12p70) production while anti-inflammatory cytokines, such as IL-10, are elevated.9C11 Multiple studies have described the SMIP004 ontogeny of murine B cell function.6 For example, murine neonatal B cell production of IL-10 suppresses pro-inflammatory responses of dendritic cells thereby inhibiting Th1-polarizing immune responses.12, 13 Immune responses can be species-specific, and relatively little is known regarding human neonatal B cell-mediated innate immune responses. Only limited data is available regarding the expression of TLRs on human neonatal B cells, and regarding the responsiveness of human neonatal B cells to TLR agonists. Two studies have reported that human cord blood B cells respond to CpG stimulation by producing cytokines and up-regulating genes that are involved in plasma cell differentiation.14, 15 Because TLR agonists may be used as vaccine adjuvants and characterization of age-specific TLR function may inform adjuvanted vaccine development, we sought to more completely characterize the expression of SMIP004 TLRs in human circulating B cells and B cell subsets from both newborn and adult subjects, and to evaluate TLR- mediated cytokine production and Ig class-switch recombination by circulating newborn (cord blood) and adult (peripheral blood) B cells. To characterize B cell ontogeny, our approach entailed comparing newborn and adult total na?ve B cells, as this subset represents the vast majority of all neonatal B cells,16 and has been the subject of prior studies in this area.17C19 We have found that neonatal na?ve B cells exhibit distinct functional expression of TLRs with relatively high TLR9 expression and TLR9-mediated cytokine induction and impaired CD40-mediated responses, including Ig production and class switching, providing fresh insights into the ontogeny and innate function of this key leukocyte population. Materials and Methods Blood collection Peripheral blood was collected after informed consent from healthy adult volunteers according to Boston Childrens Hospital Institutional Review Board-approved protocols (Boston, MA, USA; mean age 32.4 years) and newborn cord blood (mean gestational age 39.1 weeks) was collected immediately after elective cesarean section delivery (epidural anesthesia) of the placenta. Births to HIV-positive mothers were excluded. Human experimentation guidelines of the US Department of Health and Human Services, the Brigham and Womens Hospital, Beth Israel Medical Center, and Boston Childrens Hospital were observed, following protocols approved by the local institutional review boards. Number of repeats (n) indicates the number of independent experiments. No subject was studied more.