IL-21-producing effector Tfh cells promote B cell alloimmunity in lymph nodes and kidney allografts.

Follicular helper T (Tfh) cells have been implicated in controlling rejection after allogeneic kidney transplantation, but the precise subsets, origins, and functions of Tfh cells in this process have not been fully characterized. Here we show that a subset of effector Tfh cells marked by previous IL-21 production is potently induced during allogeneic kidney transplantation and is inhibited by immunosuppressive agents. Single-cell RNA-Seq revealed that these lymph node (LN) effector Tfh cells have transcriptional and clonal overlap with IL-21-producing kidney-infiltrating Tfh cells, implicating common origins and developmental trajectories. To investigate the precise functions of IL-21-producing effector Tfh cells in LNs and allografts, we used a mouse model to selectively eliminate these cells and assessed allogeneic B cell clonal dynamics using a single B cell culture system. We found that IL-21-producing effector Tfh cells were essential for transplant rejection by regulating donor-specific germinal center B cell clonal dynamics both systemically in the draining LN and locally within kidney grafts. Thus, IL-21-producing effector Tfh cells have multifaceted roles in Ab-mediated rejection after kidney transplantation by promoting B cell alloimmunity.

MHC Tetramers Specifically Identify High- and Low-avidity Donor-specific B Cells in Transplantation Tolerance and Rejection.

BACKGROUND: Although donor-specific antibody pre- and posttransplantation is routinely assessed, accurate quantification of memory alloreactive B cells that mediate recall antibody response remains challenging. Major histocompatibility complex (MHC) tetramers have been used to identify alloreactive B cells in mice and humans, but the specificity of this approach has not been rigorously assessed. METHODS: B-cell receptors from MHC tetramer-binding single B cells were expressed as mouse recombinant immunoglobulin G1 (rIgG1) monoclonal antibodies, and the specificity was assessed with a multiplex bead assay. Relative binding avidity of rIgG1 was measured by modified dilution series technique and surface plasmon resonance. Additionally, immunoglobulin heavy chain variable regions of 50 individual B-cell receptors were sequenced to analyze the rate of somatic hypermutation. RESULTS: The multiplex bead assay confirmed that expressed rIgG1 monoclonal antibodies were preferentially bound to bait MHC class II I-E d over control I-A d and I-A b tetramers. Furthermore, the dissociation constant 50 binding avidities of the rIgG1 ranged from 10 mM to 7 nM. The majority of tetramer-binding B cells were low avidity, and ~12.8% to 15.2% from naive and tolerant mice and 30.9% from acute rejecting mice were higher avidity (dissociation constant 50 <1 mM). CONCLUSIONS: Collectively, these studies demonstrate that donor MHC tetramers, under stringent binding conditions with decoy self-MHC tetramers, can specifically identify a broad repertoire of donor-specific B cells under conditions of rejection and tolerance.

Beyond Adaptive Alloreactivity: Contribution of Innate B Cells to Allograft Inflammation and Rejection.

Innate B cells are a heterogeneous group of cells that function in maintaining homeostatic levels of circulating natural antibodies and being the first line of defense against infections. Innate B-1 cells and marginal zone B cells may relocate to lymphoid follicles and differentiate into cytokine and antibody-secreting cells in T-independent and T-dependent manners. Although marginal zone B cells are widely described in humans, the presence of B-1 cells is more controversial. Here, we review the basic features of the innate B-cell subsets identified in mice and their equivalent in humans, as well as their potential roles in transplantation. We summarize the findings of Cascalho and colleagues on the unexpected protective role of tumor necrosis factor receptor superfamily member 13B in regulating circulating levels of protective natural immunoglobulin M, and the studies by Zorn and colleagues on the potential pathogenic role for polyreactive innate B cells infiltrating allograft explants. Finally, we discuss our studies that took a transcriptomic approach to identify innate B cells infiltrating kidney allografts with antibody-mediated rejection and to demonstrate that local antigens within the allograft together with inflammation may induce a loss of B-cell tolerance.

Differential CD4+ T-Cell Cytokine and Cytotoxic Responses Between Reactivation and Latent Phases of Herpes Zoster Infection.

Background: CD4+ T cells are a critical component of effective immune responses to varicella zoster virus (VZV), but their functional properties during the reactivation acute vs latent phase of infection remain poorly defined. Methods: Here we assessed the functional and transcriptomic properties of peripheral blood CD4+ T cells in persons with acute herpes zoster (HZ) compared to those with a prior history of HZ infection using multicolor flow cytometry and RNA sequencing. Results: We found significant differences between the polyfunctionality of VZV-specific total memory, effector memory, and central memory CD4+ T cells in acute vs prior HZ. VZV-specific CD4+ memory T-cell responses in acute HZ reactivation had higher frequencies of IFN-γ and IL-2 producing cells compared to those with prior HZ. In addition, cytotoxic markers were higher in VZV-specific CD4+ T cells than non-VZV-specific cells. Transcriptomic analysis of ex vivo total memory CD4+ T cells from these individuals showed differential regulation of T-cell survival and differentiation pathways, including TCR, cytotoxic T lymphocytes (CTL), T helper, inflammation, and MTOR signaling pathways. These gene signatures correlated with the frequency of IFN-γ and IL-2 producing cells responding to VZV. Conclusions: In summary, VZV-specific CD4+ T cells from acute HZ individuals had unique functional and transcriptomic features, and VZV-specific CD4+ T cells as a group had a higher expression of cytotoxic molecules including Perforin, Granzyme-B, and CD107a.

Adjuvant effect of type I interferon induced by many but not all commercial influenza vaccines.

BACKGROUND: Seasonal influenza vaccines approved and offered in the United States have varying reported degrees of effectiveness year over year and between manufacturers. Influenza vaccines produced from live virus may include single stranded RNA (ssRNA) that is a potent activator of the innate Toll-like receptor 7 (TLR-7) ligand. Plasmacytoid dendritic cells (pDC) can be activated by ssRNA to produce type I interferons such as IFN-α, which has been shown to have an adjuvant-like effect. OBJECTIVE: Our aim was to determine if IFN-α induction in peripheral blood mononuclear cells (PBMCs) exposed to eight different commercial influenza vaccines is a pDC-dependent process mediated through TLR-7 signaling. RESULTS: We demonstrate the ability of multiple vaccines to induce IFN-α in a TLR-7-dependent fashion. A number of vaccines however lacked IFN-α induction. The significance of these differences between vaccines is unclear, since all the approved vaccine formulations offer some degree of protection.

T follicular helper cells in human efferent lymph retain lymphoid characteristics.

T follicular helper cells (Tfh), a subset of CD4+ T cells, provide requisite help to B cells in the germinal centers (GC) of lymphoid tissue. GC Tfh are identified by high expression of the chemokine receptor CXCR5 and the inhibitory molecule PD-1. Although more accessible, blood contains lower frequencies of CXCR5+ and PD-1+ cells that have been termed circulating Tfh (cTfh). However, it remains unclear whether GC Tfh exit lymphoid tissues and populate this cTfh pool. To examine exiting cells, we assessed the phenotype of Tfh present within the major conduit of efferent lymph from lymphoid tissues into blood, the human thoracic duct. Unlike what was found in blood, we consistently identified a CXCR5-bright PD-1-bright (CXCR5BrPD-1Br) Tfh population in thoracic duct lymph (TDL). These CXCR5BrPD-1Br TDL Tfh shared phenotypic and transcriptional similarities with GC Tfh. Moreover, components of the epigenetic profile of GC Tfh could be detected in CXCR5BrPD-1Br TDL Tfh and the transcriptional imprint of this epigenetic signature was enriched in an activated cTfh subset known to contain vaccine-responding cells. Together with data showing shared TCR sequences between the CXCR5BrPD-1Br TDL Tfh and cTfh, these studies identify a population in TDL as a circulatory intermediate connecting the biology of Tfh in blood to Tfh in lymphoid tissue.

Regulatory roles of IL-10-producing human follicular T cells.

Mucosal lymphoid tissues such as human tonsil are colonized by bacteria and exposed to ingested and inhaled antigens, requiring tight regulation of immune responses. Antibody responses are regulated by follicular helper T (TFH) cells and FOXP3+ follicular regulatory T (TFR) cells. Here we describe a subset of human tonsillar follicular T cells identified by expression of TFH markers and CD25 that are the main source of follicular T (TF) cell-derived IL-10. Despite lack of FOXP3 expression, CD25+ TF cells resemble T reg cells in high CTLA4 expression, low IL-2 production, and their ability to repress T cell proliferation. CD25+ TF cell-derived IL-10 dampens induction of B cell class-switching to IgE. In children, circulating total IgE titers were inversely correlated with the frequencies of tonsil CD25+ TF cells and IL-10-producing TF cells but not with total T reg cells, TFR, or IL-10-producing T cells. Thus, CD25+ TF cells emerge as a subset with unique T and B cell regulatory activities that may help prevent atopy.

Spatial distribution and function of T follicular regulatory cells in human lymph nodes.

T follicular regulatory (Tfr) cells are a population of CD4+ T cells that express regulatory T cell markers and have been shown to suppress humoral immunity. However, the precise mechanisms and location of Tfr-mediated suppression in the lymph node (LN) microenvironment are unknown. Using highly multiplexed quantitative imaging and functional assays, we examined the spatial distribution, suppressive function, and preferred interacting partners of Tfr cells in human mesenteric LNs. We find that the majority of Tfr cells express low levels of PD-1 and reside at the border between the T cell zone and B cell follicle, with very few found in the germinal centers (GCs). Although PD-1+ Tfr cells expressed higher levels of CD38, CTLA-4, and GARP than PD-1Neg Tfr cells, both potently suppressed antibody production in vitro. These findings highlight the phenotypic diversity of human Tfr cells and suggest that Tfr-mediated suppression is most efficient at the T-B border and within the follicle, not in the GC.

Immune Activation at Sites of HIV/TB Co-Infection Contributes to the Pathogenesis of HIV-1 Disease.

Systemic immune activation is critical to the pathogenesis of HIV-1 disease, and is accentuated in HIV/TB co-infected patients. The contribution of immune activation at sites of HIV/TB co-infection to viral activity, CD4 T cell count, and productive HIV-1 infection remain unclear. In this study, we measured markers of immune activation both in pleural fluid and plasma, and in T cells in pleural fluid mononuclear cell (PFMC) and peripheral blood mononuclear cell (PBMC) in HIV/TB co-infected subjects. The relationship between soluble and T cell activation markers with viral load in pleural fluid and blood CD4 T cell count were assessed. The T cell phenotype and activation status of HIV-1 p24 + T cells in PFMC and PBMC from HIV/TB patients were determined. We found that T cell and macrophage-specific and non-specific soluble markers of immune activation, sCD27, sCD163, IL1Ra, and sCD14, were higher in pleural fluid as compared to plasma from HIV/TB co-infected subjects, and higher as compared to pleural fluid from TB mono-infected subjects. Intestinal fatty acid-binding protein, a marker of intestinal tract damage, in plasma from HIV/TB co-infected patients was not different than that in HIV+ subjects. Expression of HLADR and CD38 double positive (HLADR/CD38) on CD4 T cells, and CD69+ on CD8 T cells correlated with pleural fluid viral load, and inversely with blood CD4 T cell count. Higher expression of HLADR/CD38 and CCR5 on CD4 T cells, and HLADR/CD38 and CD69 on CD8 T cells in PFMC were limited to effector memory populations. HIV-1 p24+ CD8 negative (includes CD4 + and double negative T cells) effector memory T cells in PFMC had higher expression of HLADR/CD38, Ki67, and CCR5 compared to HIV-1 p24- CD8 negative PFMC. Cumulatively, these data indicate that sites of HIV/TB co-infection are the source of intense immune activation.

Micro and Nano-Scale Technologies for Cell Mechanics.

Cell mechanics is a multidisciplinary field that bridges cell biology, fundamental mechanics, and micro and nanotechnology, which synergize to help us better understand the intricacies and the complex nature of cells in their native environment. With recent advances in nanotechnology, microfabrication methods and micro-electro-mechanical-systems (MEMS), we are now well situated to tap into the complex micro world of cells. The field that brings biology and MEMS together is known as Biological MEMS (BioMEMS). BioMEMS take advantage of systematic design and fabrication methods to create platforms that allow us to study cells like never before. These new technologies have been rapidly advancing the study of cell mechanics. This review article provides a succinct overview of cell mechanics and comprehensively surveys micro and nano-scale technologies that have been specifically developed for and are relevant to the mechanics of cells. Here we focus on micro and nano-scale technologies, and their applications in biology and medicine, including imaging, single cell analysis, cancer cell mechanics, organ-on-a-chip systems, pathogen detection, implantable devices, neuroscience and neurophysiology. We also provide a perspective on the future directions and challenges of technologies that relate to the mechanics of cells.

Connective tissue and its growth factor CTGF distinguish the morphometric and molecular remodeling of the bladder in a model of neurogenic bladder.

We previously reported that mice with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS), develop profound urinary bladder dysfunction. Because neurogenic bladder in MS patients causes marked bladder remodeling, we next examined morphometric and molecular alterations of the bladder in EAE mice. EAE was created in female SJL/J mice by immunization with the p139-151 encephalitogenic peptide of myelin proteolipid protein in complete Freund's adjuvant, along with intraperitoneal injections of Bordetella pertussis toxin. Seventy days after immunization, mice were scored for the level of neurological impairment and then killed. Spinal cord sections were assessed for demyelination, inflammation, and T cell infiltration; the composition of the bladder tissue was measured quantitatively; and gene expression of markers of tissue remodeling and fibrosis was assessed. A significant increase in the bladder weight-to-body weight ratio was observed with increasing neurological impairment, and morphometric analysis showed marked bladder remodeling with increased luminal area and tissue hypertrophy. Despite increased amounts of all tissue components (urothelium, smooth muscle, and connective tissue), the ratio of connective tissue to muscle increased significantly in EAE mice compared with control mice. Marked increases in mRNA expression of collagen type I α(2), tropoelastin, transforming growth factor-ß3, and connective tissue growth factor (CTGF) were observed in EAE mice, as were decreased levels of mRNAs for smooth muscle myosin heavy chain, nerve growth factors, and muscarinic and purinergic receptors. Our results suggest that bladder remodeling corresponding to EAE severity may be due to enhanced expression of CTGF and increased growth of connective tissue.

Characterization of yeast species using surface-enhanced Raman scattering.

Surface-enhanced Raman scattering (SERS) is used for the characterization of six yeast species and six isolates. The sample for SERS analysis is prepared by mixing the yeast cells with a four times concentrated silver colloidal suspension. The scanning electron microscopy (SEM) images show that the strength of the interaction between silver nanoparticles and the yeast cells depends on the biochemical structure of the cell wall. The SERS spectra are used to identify the biochemical structures on the yeast cell wall. It is found that the density of -SH and -NH2 groups might be higher on certain yeast cell walls. Finally, the obtained SERS spectra from yeast is used for the classification of the yeast.