Ena/VASP Protein-Mediated Actin Polymerization Contributes to Naïve CD8+ T Cell Activation and Expansion by Promoting T Cell-APC Interactions In Vivo.

Naïve T cell activation in secondary lymphoid organs such as lymph nodes (LNs) occurs upon recognition of cognate antigen presented by antigen presenting cells (APCs). T cell activation requires cytoskeleton rearrangement and sustained interactions with APCs. Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are a family of cytoskeletal effector proteins responsible for actin polymerization and are frequently found at the leading edge of motile cells. Ena/VASP proteins have been implicated in motility and adhesion in various cell types, but their role in primary T cell interstitial motility and activation has not been explored. Our goal was to determine the contribution of Ena/VASP proteins to T cell-APC interactions, T cell activation, and T cell expansion in vivo. Our results showed that naïve T cells from Ena/VASP-deficient mice have a significant reduction in antigen-specific T cell accumulation following Listeria monocytogenes infection. The kinetics of T cell expansion impairment were further confirmed in Ena/VASP-deficient T cells stimulated via dendritic cell immunization. To investigate the cause of this T cell expansion defect, we analyzed T cell-APC interactions in vivo by two-photon microscopy and observed fewer Ena/VASP-deficient naïve T cells interacting with APCs in LNs during priming. We also determined that Ena/VASP-deficient T cells formed conjugates with significantly less actin polymerization at the T cell-APC synapse, and that these conjugates were less stable than their WT counterparts. Finally, we found that Ena/VASP-deficient T cells have less LFA-1 polarized to the T cell-APC synapse. Thus, we conclude that Ena/VASP proteins contribute to T cell actin remodeling during T cell-APC interactions, which promotes the initiation of stable T cell conjugates during APC scanning. Therefore, Ena/VASP proteins are required for efficient activation and expansion of T cells in vivo.

Elevated Detection of Dual Antibody B Cells Identifies Lupus Patients With B Cell-Reactive VH4-34 Autoantibodies.

About 5% of B cells in healthy mice and humans are allelically or isotypically included and hence co-express two different antibodies. In mice, dual antibody B cells (B2R) expand with systemic autoimmunity, co-express autoreactive and non-autoreactive antibodies, and participate in immune responses, but this phenomenon is strain dependent. This study was developed with two goals: 1) to establish the contribution of TLR and IFN receptor signaling to the development of germinal center B cells that express two antibodies in MRL/lpr mice; and 2) to determine whether B2R B cells are increased and particularly activated in a subset of adult patients diagnosed with systemic lupus erythematosus (SLE). Results from the MRL/lpr studies indicate that the enhanced differentiation of dual-κ B cells into germinal center B cells is due to a heightened response to TLR7 and TLR9 signaling, further fueled by an increased response to type II IFN. To understand the clinical and translational implications of our observations in mouse B2R B cells, cohorts of SLE patients and healthy controls were recruited and evaluated for expression of dual BCRs. Results from flow cytometry and microscopy revealed supraphysiological frequencies of κ+λ+ B2R cells in one fourth of the SLE patients. Abnormal numbers of κ+λ+ B cells correlated with higher frequencies of activated naïve B cells and age-associated B cells, and a lower proportion of "B cells that are naïve IgD+" (BND). However, results from single cell V(D)J sequencing demonstrated that these high κ+λ+ SLE patients harbored normal frequencies of κ+λ+ and other B2R B cells. and we further show that their B cells were instead decorated by κ and λ VH4-34 autoantibodies. Thus, our findings indicate that elevated flow cytometric detection of isotypically-included B cells can identify patients with high titers of B cell-reactive VH4-34 autoantibodies and abnormal distribution of B cell subsets relevant to autoimmunity.

Hematopoietic stem cells produce intermediate lineage adipocyte progenitors that simultaneously express both myeloid and mesenchymal lineage markers in adipose tissue.

Some adipocytes are produced from bone marrow hematopoietic stem cells. In vitro studies previously indicated that these bone marrow-derived adipocytes (BMDAs) were generated from adipose tissue macrophage (ATM) that lose their hematopoietic markers and acquire mesenchymal markers prior to terminal adipogenic differentiation. Here we interrogated whether this hematopoietic-to-mesenchymal transition drives BMDA production In vitro. We generated transgenic mice in which the lysozyme gene promoter (LysM) indelibly labeled ATM with green fluorescent protein (GFP). We discovered that adipose stroma contained a population of LysM-positive myeloid cells that simultaneously expressed hematopoietic/myeloid markers (CD45 and CD11b), and mesenchymal markers (CD29, PDGFRa and Sca-1) typically found on conventional adipocyte progenitors. These cells were capable of adipogenic differentiation In vitro and In vitro, while other stromal populations deficient in PDGFRa and Sca-1 were non-adipogenic. BMDAs and conventional adipocytes expressed common fat cell markers but exhibited little or no expression of hematopoietic and mesenchymal progenitor cell markers. The data indicate that BMDAs are produced from ATM simultaneously expressing hematopoietic and mesenchymal markers rather than via a stepwise hematopoietic-to-mesenchymal transition. Because BMDA production is stimulated by high fat feeding, their production from hematopoietic progenitors may maintain adipocyte production when conventional adipocyte precursors are diminished.

Impact of HIV-1 Infection and Antigen Class on T Follicular Helper Cell Responses to Pneumococcal Polysaccharide-Protein Conjugate Vaccine-13.

Pneumococcal infections are common and serious complications of HIV-1 disease. Prevention has been compromised by the limited magnitude and quality of Ab responses to T cell-independent type 2 pneumococcal capsular polysaccharides (PPS). The pneumococcal polysaccharide-protein conjugate vaccine-13 (PCV-13) contains PPS conjugated to the T cell-dependent protein (diphtheria toxoid [DT] [CRM197]). We investigated the differential response to PPS and DT by human Ab-secreting B cells (ASC) after immunization with PCV-13 in newly diagnosed healthy HIV+ and control adults. The numbers of PPS-specific IgG ASC increased significantly and similarly in HIV+ and controls. However, DT-specific IgG ASC increased in controls but not HIV+ subjects. To determine the cellular basis of these disparate responses to DT and PPS, we characterized the frequency and activation of T follicular helper (Tfh) cells, the predominant T cell subset providing B cell help. Expression of inducible T cell costimulator (ICOS), which sustains Tfh function and phenotype, increased significantly among controls, when compared with the HIV+ group. Increases in ICOS+ Tfh correlated with changes in T-dependent, DT-specific IgG ASC in controls but not in HIV+ In contrast, ICOS expression did not correlate with T cell-independent type 2 PPS-specific ASC in either group. Of note, upon optimized ex vivo stimulation, CD4 T cells from HIV+ subjects differentiated into Tfh cells and formed synapses with Raji B cells at frequencies similar to that of controls. In summary, PCV-13-induced increase in ICOS expression on Tfh was associated with responses to DT, which was compromised in recently diagnosed healthy HIV+ adults and can be restored ex vivo by providing effective Tfh-differentiating signals.

The Impact of Moderate or High-Intensity Combined Exercise on Systemic Inflammation Among Older Persons With and Without HIV.

BACKGROUND: We investigated whether higher-intensity exercise provided greater decrease in markers of inflammation, and whether responses differed by HIV serostatus. METHODS: People with HIV (PWH; n = 32) and controls (n = 37) aged 50-75 years completed 12 weeks moderate-intensity exercise, then were randomized to moderate- or high-intensity exercise for 12 additional weeks (n = 27 and 29, respectively). Inflammation biomarkers were measured at 0, 12, 24 weeks. Mixed and multiple regression models were adjusted for baseline inflammation, age, and body mass index. RESULTS: Baseline tumor necrosis factor-α (TNF-α), soluble TNF receptor 2 (sTNFR2), and soluble CD14 (sCD14) were significantly higher among PWH than controls (P < .04). From week 0-12, changes in interleukin-6 (IL-6), TNF-α, and sTNFR1 were not significantly different by HIV serostatus. We found no significant interaction between HIV serostatus/exercise intensity on week 12-24 changes in IL-6, TNF-α, and sTNFR1. Among high-intensity exercisers, PWH and controls had significant increases in sCD14 (P ≤ .003), controls significant increases in IL-10 (P = .01), and PWH nonsignificant decrease in highly sensitive C-reactive protein (P = .07). Other markers were not significantly different by serostatus or intensity. CONCLUSIONS: Moderate and high-intensity exercise elicited similar effects on inflammation among PWH and controls, with additional beneficial effects seen among high-intensity exercisers. Increase in sCD14 and attenuated IL-10 increase (PWH only) merit further study. CLINICAL TRIALS REGISTRATION: NCT02404792.

Streptococcus pneumoniae G5 domains bind different ligands.

Many bacterial pathogens express small G5 domains that exist in the context of various membrane-anchored proteins and these G5 domains have been associated with colonization, cellular adhesion, and biofilm formation. However, despite over a decade since the computational prediction of these G5 domains, many remain uncharacterized, particularly those from Streptococcus pneumoniae. Of five previously predicted G5 domains we found that four of these, all derived from S. pneumoniae, are independently folded modules. As one of these exhibits extreme line broadening due to self-association, we were able to use NMR solution studies to probe the potential ligand interactions of the remaining three G5 domains. None of these G5 domains engage N-acetylglucosamine (NAG) as previously predicted but do interact with other small molecules that may modulate adherence to both bacteria and host cells. Specifically, while all G5 domains tested engage Zn, only one of these G5 domains engage heparin. NMR solution structural studies of the IgA1 Protease G5 (IgA1P-G5) and endo-beta-N-acetylglucosaminidase-D G5 (ENDD-G5) also facilitated identification of the ligand binding sites and confirm the typical G5 fold that comprises two connected ß-sheets with no canonical core. NMR relaxation experiments indicate flexibility on both ends and within the connecting regions between the ß-sheets. Our studies thus establish a basis for future biological experiments to test whether the ligands presented here are involved in bacterial adherence, either to bacteria or to host cells.

Streptococcus pneumoniae IgA1 protease: A metalloprotease that can catalyze in a split manner in vitro.

IgA1 proteases (IgA1P) from diverse pathogenic bacteria specifically cleave human immunoglobulin A1 (IgA1) at the hinge region, thereby thwarting protective host immune responses. Streptococcus pneumoniae (S. pneumoniae) IgA1P shares no sequence conservation with serine or cysteine types of IgA1Ps or other known proteins, other than a conserved HExxH Zn-binding motif (1604-1608) found in metalloproteases. We have developed a novel expression system to produce the mature S. pneumoniae IgA1P and we have discovered that this form is both attached to the bacterial cell surface and released in its full form. Our data demonstrate that the S. pneumoniae IgA1P comprises two distinct regions that associate to form an active metalloprotease, the first such example of a metalloprotease that can be split in vitro and recombined to form an active enzyme. By capitalizing on this novel domain architecture, we show that the N-terminal region of S. pneumoniae IgA1P comprises the primary binding region for IgA1, although the C-terminal region of S. pneumoniae IgA1P is necessary for cleavage of IgA1. Our findings lend insight into the protein domain architecture of the S. pneumoniae IgA1P and function of this important virulence factor for S. pneumoniae infection.

Mother-to-Child HIV-1 Transmission Events Are Differentially Impacted by Breast Milk and Its Components from HIV-1-Infected Women.

Breast milk is a vehicle of infection and source of protection in post-natal mother-to-child HIV-1 transmission (MTCT). Understanding the mechanism by which breast milk limits vertical transmission will provide critical insight into the design of preventive and therapeutic approaches to interrupt HIV-1 mucosal transmission. However, characterization of the inhibitory activity of breast milk in human intestinal mucosa, the portal of entry in postnatal MTCT, has been constrained by the limited availability of primary mucosal target cells and tissues to recapitulate mucosal transmission ex vivo. Here, we characterized the impact of skimmed breast milk, breast milk antibodies (Igs) and non-Ig components from HIV-1-infected Ugandan women on the major events of HIV-1 mucosal transmission using primary human intestinal cells and tissues. HIV-1-specific IgG antibodies and non-Ig components in breast milk inhibited the uptake of Ugandan HIV-1 isolates by primary human intestinal epithelial cells, viral replication in and transport of HIV-1- bearing dendritic cells through the human intestinal mucosa. Breast milk HIV-1-specific IgG and IgA, as well as innate factors, blocked the uptake and transport of HIV-1 through intestinal mucosa. Thus, breast milk components have distinct and complementary effects in reducing HIV-1 uptake, transport through and replication in the intestinal mucosa and, therefore, likely contribute to preventing postnatal HIV-1 transmission. Our data suggests that a successful preventive or therapeutic approach would require multiple immune factors acting at multiple steps in the HIV-1 mucosal transmission process.

Infant morbidity, mortality, and breast milk immunologic profiles among breast-feeding HIV-infected and HIV-uninfected women in Botswana.

BACKGROUND: Infants of human immunodeficiency virus (HIV)-infected women have high mortality, but the immunologic integrity and protection afforded by the breast milk of HIV-infected women is unknown. METHODS: We determined morbidity and mortality by 24 months among breast-fed infants of 588 HIV-infected and 137 HIV-uninfected women followed-up in a clinical trial in Botswana. A matched case-control study compared clinical, behavioral, and breast milk immunologic parameters among 120 HIV-infected women by infant outcome. Breast milk factors were also compared between HIV-infected and HIV-uninfected women. RESULTS: Twenty-four-month mortality was 29.5% among HIV-infected infants, 6.7% among HIV-exposed uninfected infants, and 1.6% among HIV-unexposed infants. No differences were detected in breast milk immunologic profiles of HIV-infected women whose infants were either ill or well. Discontinuation of breast-feeding was the strongest predictor of illness (P<.001). Levels in breast milk of pathogen-specific immunoglobulin (Ig) G and IgA to Haemophilus influenzae, Campylobacter jejuni, Helicobacter pylori, Streptococcus pneumoniae, and innate immune factors were not lower among HIV-infected women than among HIV-uninfected women. CONCLUSIONS: Mortality was higher among HIV-infected and HIV-exposed infants than among HIV-unexposed infants. However, the immunologic profiles of breast milk among HIV-infected women were intact, and discontinuation of breast-feeding was the primary risk for infant morbidity. Thus, the breast milk of HIV-infected women may confer protection against common infant pathogens. TRIAL REGISTRATION: (ClinicalTrials.Gov) identifiers: NCT00197691 and NCT00197652.