Biomarkers detected in cord blood predict vaccine responses in young infants.

Introduction: Factors influencing vaccine immune priming in the first year of life involve both innate and adaptive immunity but there are gaps in understanding how these factors sustain vaccine antibody levels in healthy infants. The hypothesis was that bioprofiles associated with B cell survival best predict sustained vaccine IgG levels at one year. Methods: Longitudinal study of plasma bioprofiles in 82 term, healthy infants, who received standard recommended immunizations in the United States, with changes in 15 plasma biomarker concentrations and B cell subsets associated with germinal center development monitored at birth, soon after completion of the initial vaccine series at 6 months, and prior to the 12-month vaccinations. Post vaccination antibody IgG levels to Bordetella pertussis, tetanus toxoid, and conjugated Haemophilus influenzae type B (HiB) were outcome measures. Results: Using a least absolute shrinkage and selection operator (lasso) regression model, cord blood (CB) plasma IL-2, IL-17A, IL-31, and soluble CD14 (sCD14) were positively associated with pertussis IgG levels at 12 months, while CB plasma concentrations of APRIL and IL-33 were negatively associated. In contrast, CB concentrations of sCD14 and APRIL were positively associated with sustained tetanus IgG levels. A separate cross-sectional analysis of 18 mother/newborn pairs indicated that CB biomarkers were not due to transplacental transfer, but rather due to immune activation at the fetal/maternal interface. Elevated percentages of cord blood switched memory B cells were positively associated with 12-month HiB IgG levels. BAFF concentrations at 6 and 12 months were positively associated with pertussis and HiB IgG levels respectively. Discussion: Sustained B cell immunity is highly influenced by early life immune dynamics beginning prior to birth. The findings provide important insights into how germinal center development shapes vaccine responses in healthy infants and provide a foundation for studies of conditions that impair infant immune development.

Higher soluble CD14 levels are associated with lower visuospatial memory performance in youth with HIV.

OBJECTIVE: HIV-associated neurocognitive disorders persist despite early antiretroviral therapy (ART) and optimal viral suppression. We examined the relationship between immunopathogenesis driven by various pathways of immune activation and discrete neurocognitive performance domains in youth with HIV (YWH). DESIGN: Observational cross-sectional study. METHODS: YWH, ages 20-28 years, enrolled in Adolescent Medicine Trials Network 071/101 were assessed for biomarkers of macrophage, lymphocyte activation, and vascular inflammation using ELISA/multiplex assays. Standardized neurocognitive tests were performed, and demographically adjusted z-scores were combined to form indices of attention, motor, executive function, verbal, and visuospatial memory. Cross-sectional analysis of the relationship between 18 plasma inflammatory biomarkers and each neurocognitive domain was performed. Linear regression models were fit for each combination of log-transformed biomarker value and neurocognitive domain score, and were adjusted for demographics, socioeconomic status, substance use, depression, CD4 T-cell count, HIV viral load, and ART status. RESULTS: Study included 128 YWH [mean age 23.8 (SD 1.7) years, 86% men, 68% African American]. Verbal and visuospatial memory domains were most significantly impaired in the cohort (z = -1.59 and -1.0, respectively). Higher sCD14 was associated with impaired visuospatial memory, which remained robust after adjusting for other biomarkers, demographics, and HIV-associated covariates. Among biomarkers of vascular inflammation, sICAM-1 was negatively associated with verbal memory and attention, whereas sVCAM-1 was positively associated with executive function and visuospatial memory. Specific neurocognitive domains were not associated with sCD163, LPS, or CCL2 levels. CONCLUSION: Impaired visuospatial memory in YWH is associated with immune activation, as reflected by higher sCD14.

Tenofovir Has Minimal Effect on Biomarkers of Bone Health in Youth with HIV Receiving Initial Antiretroviral Therapy.

Both HIV infection and tenofovir disoproxil fumarate (TDF) treatment adversely impact bone metabolism and may lead to osteopenia, which has critical implications for youth with HIV (YWH). This study evaluates changes in the biomarkers of bone metabolism and inflammation among YWH receiving initial treatment with TDF- and non-TDF-containing antiretroviral therapies (ARTs). YWH [n = 23, median age 21 years (range 18-24), 87% male, 61% African American] were assessed for inflammatory and bone metabolism biomarkers at enrollment, after 48 weeks of TDF-containing ART, and 96 weeks of ART without TDF with continued viral suppression. Spearman's rank correlation evaluated biomarker associations. Bone alkaline phosphatase, parathyroid hormone, and osteopontin increased after TDF treatment. All fell after TDF was discontinued. Levels of RANKL and osteoprotegerin did not change throughout the study. There was little correlation between biomarkers of bone metabolism and either macrophage or lymphocyte activation at any time point. Our results establish baseline associations between bone metabolism and immune biomarkers for this population, and find that before CD4 T cell decline chronic inflammation does not perturb biomarkers of bone metabolism among YWH. The adverse effects of TDF on bone health may be marginal for YWH at the early stages of disease.

The Regulatory B Cell Compartment Expands Transiently During Childhood and Is Contracted in Children With Autoimmunity.

OBJECTIVE: Regulatory B cells that inhibit immune responses through interleukin-10 (IL-10) secretion (B10 cells) have been characterized in adult subjects with autoimmune disease. The aim of this study was to characterize B10 cells in individuals across the entire age range of normal human development and changes in their frequency and numbers in children with autoimmunity. METHODS: The phenotype and numbers of B10 cells in blood were examined in healthy individuals and children with autoimmunity, using flow cytometry. B10 cell function was assessed by measuring the effect of B cell-derived IL-10 on interferon-γ (IFNγ) expression by CD4+ T cells. Serum cytokine levels were measured by enzyme-linked immunosorbent assay. RESULTS: The frequency of B10 cells transiently increased during childhood, when up to 30% of B cells were competent to produce IL-10, compared with the low frequencies in healthy newborns (3-4%) and adults (7-9%). The surface phenotype of B10 cells in children revealed age-dependent variability. B10 cells from children were distinct from proinflammatory cytokine-producing B cells and down-regulated IFNγ production by CD4+ T cells in vitro. Compared with age-matched healthy controls, children with autoimmunity had lower numbers and frequencies of B10 cells (decreased by 39% and 48%, respectively), higher IFNγ levels, and lower IL-21 levels in serum. IFNγ inhibited, whereas IL-21 promoted, B cell IL-10 competence in vitro. CONCLUSION: B10 cells, a functionally defined cell subset with a variable surface phenotype reflective of overall B cell development, transiently expand during childhood. B10 cell frequencies and numbers were decreased in children with autoimmunity, which may be explained in part by alterations in serum IFNγ and IL-21 that differentially regulate B10 cell development.

A reevaluation of CD22 expression in human lung cancer.

CD22 is a transmembrane glycoprotein expressed by mature B cells. It inhibits signal transduction by the B-cell receptor and its coreceptor CD19. Recent reports indicate that most human lung cancer cells and cell lines express CD22, making it an important new therapeutic target for lung cancer. The objective of our studies was to independently validate these results with the goal of testing the efficacy of our CD22 immunotoxins on lung cancer cell lines. As determined by quantitative real-time PCR analysis, we found that levels of CD22 mRNA in a panel of human lung cancer cell lines were 200 to 60,000-fold lower than those observed in the human CD22(+) Burkitt lymphoma cells, Daudi. Using flow cytometry with a panel of CD22 monoclonal antibodies and Western blot analyses, we could not detect surface or intracellular expression of CD22 protein in a panel of lung cancer cell lines. In addition, the in vitro proliferation of the lung tumor cell lines was not affected by either CD22 antibodies or our highly potent anti-CD22 immunotoxin. In contrast, CD22(+) Daudi cells expressed high levels of CD22 mRNA and protein, and were sensitive to our CD22 immunotoxin. Importantly, primary non-small cell lung cancers from more than 250 patient specimens did not express detectable levels of CD22 protein as assessed by immunohistochemistry. We conclude that CD22 is not expressed at measurable levels on the surface of lung cancer cells, and that these cells cannot be killed by anti-CD22 immunotoxins.

Regulatory B cell (B10 Cell) expansion during Listeria infection governs innate and cellular immune responses in mice.

Pathogens use numerous methods to subvert host immune responses, including the modulation of host IL-10 production by diverse cell types. However, the B cell sources of IL-10 and their overall influence on innate and cellular immune responses have not been well characterized during infections. Using Listeria as a model pathogen, infection drove the acute expansion of a small subset of regulatory B cells (B10 cells) that potently suppress inflammation and autoimmunity through the production of IL-10. Unexpectedly, spleen bacteria loads were 92-97% lower in B10 cell-deficient CD19(-/-) mice, in mice depleted of mature B cells, and in mice treated with CD22 mAb to preferentially deplete B10 cells before infection. By contrast, the adoptive transfer of wild-type B10 cells reduced bacterial clearance by 38-fold in CD19(-/-) mice through IL-10-dependent pathways. B10 cell depletion using CD22 mAb significantly enhanced macrophage phagocytosis of Listeria and their production of IFN-γ, TNF-α, and NO ex vivo. Accelerated bacteria clearance following B10 cell depletion significantly reduced Ag-specific CD4(+) T cell proliferation and cytokine production, but did not alter CD8(+) T cell responses. B10 cell regulatory function during innate immune responses was nonetheless dependent on cognate interactions with CD4(+) T cells because B10 cells deficient in IL-10, MHC-II, or IL-21R expression did not influence Listeria clearance. Thus, Listeria manipulates immune responses through a strategy of immune evasion that involves the preferential expansion of endogenous B10 cells that regulate the magnitude and duration of both innate and cellular immune responses.

Characterization of a rare IL-10-competent B-cell subset in humans that parallels mouse regulatory B10 cells.

Regulatory B cells control inflammation and autoimmunity in mice, including the recently identified IL-10-competent B10 cell subset that represents 1% to 3% of spleen B cells. In this study, a comparable IL-10-competent B10 cell subset was characterized in human blood. B10 cells were functionally identified by their ability to express cytoplasmic IL-10 after 5 hours of ex vivo stimulation, whereas progenitor B10 (B10pro) cells required 48 hours of in vitro stimulation before they acquired the ability to express IL-10. B10 and B10pro cells represented 0.6% and approximately 5% of blood B cells, respectively. Ex vivo B10 and B10pro cells were predominantly found within the CD24(hi)CD27(+) B-cell subpopulation that was able to negatively regulate monocyte cytokine production through IL-10-dependent pathways during in vitro functional assays. Blood B10 cells were present in 91 patients with rheumatoid arthritis, systemic lupus erythematosus, primary Sjögren syndrome, autoimmune vesiculobullous skin disease, or multiple sclerosis, and were expanded in some cases as occurs in mice with autoimmune disease. Mean B10 + B10pro-cell frequencies were also significantly higher in patients with autoimmune disease compared with healthy controls. The characterization of human B10 cells will facilitate their identification and the study of their regulatory activities during human disease.

Therapeutic B cell depletion impairs adaptive and autoreactive CD4+ T cell activation in mice.

CD20 antibody depletion of B lymphocytes effectively ameliorates multiple T cell-mediated autoimmune diseases through mechanisms that remain unclear. To address this, a mouse CD20 antibody that depletes >95% of mature B cells in mice with otherwise intact immune systems was used to assess the role of B cells in CD4(+) and CD8(+) T cell activation and expansion in vivo. B cell depletion had no direct effect on T cell subsets or the activation status of CD4(+) and CD8(+) T cells in naive mice. However, B cell depletion impaired CD4(+) T cell activation and clonal expansion in response to protein antigens and pathogen challenge, whereas CD8(+) T cell activation was not affected. In vivo dendritic cell ablation, along with CD20 immunotherapy, revealed that optimal antigen-specific CD4(+) T cell priming required both B cells and dendritic cells. Most importantly, B cell depletion inhibited antigen-specific CD4(+) T cell expansion in both collagen-induced arthritis and autoimmune diabetes mouse models. These results provide direct evidence that B cells contribute to T cell activation and expansion in vivo and offer insights into the mechanism of action for B cell depletion therapy in the treatment of autoimmunity.

B cell depletion delays collagen-induced arthritis in mice: arthritis induction requires synergy between humoral and cell-mediated immunity.

Rheumatoid arthritis is a systemic autoimmune disease. B cells are likely to play a critical role in arthritis pathogenesis, although it is unclear whether they are necessary for disease induction, autoantibody production, or disease progression. To assess the role of B cells in inflammatory arthritis, B cells were depleted using mouse anti-mouse CD20 mAbs in a mouse model of collagen-induced arthritis. CD20 mAbs effectively depleted mature B cells from adult DBA-1 mice. When B cells were depleted using CD20 mAbs before collagen immunization, there was a delay in disease onset and autoantibody production, with significantly diminished severity of arthritis both clinically and histologically. B cell depletion further delayed disease onset if initiated before, as well as after, collagen immunization. However, in both cases, the eventual reappearance of peripheral B cells triggered autoantibody production and the subsequent development of arthritis in collagen-sensitized mice. By contrast, B cell depletion after collagen immunizations did not have a significant effect on arthritis progression or severity. Thus, disease symptoms were only induced when peripheral B cells and their autoantibody products were present in collagen-immunized mice, documenting a critical role for B cells during the elicitation phase of collagen-induced arthritis. These studies suggest that B cell depletion strategies will be most effective when initiated early in the development of inflammatory arthritis, with sustained B cell depletion required to inhibit the production of isotype-switched pathogenic Abs and the evolution of joint inflammation and destruction.

CD25+CD4+ regulatory T cell migration requires L-selectin expression: L-selectin transcriptional regulation balances constitutive receptor turnover.

The molecular mechanisms controlling regulatory CD25(+)Foxp3(+)CD4(+) T cell (T(reg)) migration are central to in vivo immune responses. T(reg) cell subsets differentially express L-selectin, an adhesion molecule mediating lymphocyte migration to peripheral LNs (PLNs) and leukocyte rolling during inflammation. In this study, L-selectin was essential for T(reg) cell migration and normal tissue distribution. Specifically, there was a 90% reduction in PLN T(reg) cells in L-selectin(-/-) mice with a compensatory increase in spleen T(reg) cell numbers. Unexpectedly, however, 40% of the CD4(+) T cells remaining within PLNs of L-selectin(-/-) mice were T(reg) cells. The migratory properties of T(reg) cells were nonetheless markedly different from those of naive CD4(+) T cells, with 3- to 9-fold lower migration of T(reg) cells into PLNs and approximately 2-fold lower migration into the spleen. T(reg) cells also turned over cell surface L-selectin at a faster rate than CD25(-)CD4(+) T cells, but maintained physiologically appropriate L-selectin densities for optimal migration. Specifically, T(reg) cells expressed 30-40% more cell surface L-selectin when its endoproteolytic cleavage was blocked genetically, which resulted in a 2-fold increase in T(reg) cell migration into PLNs. However, increased L-selectin cleavage by T(reg) cells in wild-type mice was accompanied by 2-fold higher L-selectin mRNA levels, which resulted in equivalent cell surface L-selectin densities on T(reg) and naive T cells. Thus, T(reg) cells and CD25(-)CD4(+) T cells share similar requirements for L-selectin expression during migration, although additional molecular mechanisms constrain T(reg) cell migration beyond what is required for naive CD4(+) T cell migration.

The peritoneal cavity provides a protective niche for B1 and conventional B lymphocytes during anti-CD20 immunotherapy in mice.

Although anti-CD20 immunotherapy effectively treats human lymphoma and autoimmune disease, the in vivo effect of immunotherapy on tissue B cells and their subsets is generally unknown. To address this, anti-mouse CD20 mAbs were used in a mouse model in which the extent and kinetics of tissue B cell depletion could be assessed in vivo. CD20 mAb treatment depleted most mature B cells within 2 days, with 95-98% of B cells in the bone marrow, blood, spleen, lymph nodes, and gut-associated lymphoid tissues depleted by day 7, including marginal zone and follicular B cells. The few spleen B cells remaining after CD20 mAb treatment included pre-B, immature, transitional, and some B1 B cells that expressed CD20 at low levels. By contrast, peritoneal cavity B cells expressed normal CD20 densities and were coated with CD20 mAb, but only 30-43% of B1 cells and 43-78% of B2 cells were depleted by day 7. Spleen B cells adoptively transferred into the peritoneal cavity were similarly resistant to mAb-induced depletion, while transferred B cells that had migrated to the spleen were depleted. However, peritoneal B1 and B2 cells were effectively depleted in mAb-treated wild-type and C3-deficient mice by thioglycolate-induced monocyte migration into this otherwise privileged niche. Inflammation-elicited effector cells did not promote peritoneal cavity B cell depletion in FcR-deficient mice treated with CD20 mAb. Thus, the majority of CD20(+) cells and B cell subsets within lymphoid tissues and the peritoneum could be depleted efficiently in vivo through Fc-dependent, but C-independent pathways during anti-CD20 immunotherapy.

A new twist to the leukocyte adhesion cascade: intimate cooperation is key.

Leukocyte migration from blood into lymphoid and non-lymphoid tissues requires a highly orchestrated series of adhesive and signaling events. A network of adhesion molecules with overlapping functions mediate the capture, rolling and firm adhesion of leukocytes to the vascular endothelium. A new study adds a novel twist to this paradigm by demonstrating that two adhesion molecules, CD44 and VLA-4, must be physically associated with each other on activated T cells to mediate efficient rolling and firm adhesion.

Leukocyte migration is regulated by L-selectin endoproteolytic release.

L-selectin mediates lymphocyte migration to peripheral lymph nodes and leukocyte rolling on vascular endothelium during inflammation. One unique feature that distinguishes L-selectin from other adhesion molecules is that it is rapidly cleaved from the cell surface after cellular activation. The biological significance of L-selectin endoproteolytic release was determined by generating gene-targeted mice expressing a modified receptor that was not cleaved from the cell surface. Blocking L-selectin cleavage on antigen-stimulated lymphocytes allowed their continued migration to peripheral lymph nodes and inhibited their short-term redirection to the spleen. Blocking homeostatic L-selectin cleavage also resulted in a constitutive 2-fold increase in overall L-selectin expression by leukocytes. As a result, neutrophils entered the inflamed peritoneum in greater numbers or for a longer duration. Thus, endoproteolytic cleavage regulates both homeostatic and activation-induced changes in cell surface L-selectin density, which directs the migration patterns of activated lymphocytes and neutrophils in vivo.

Leukocyte rolling velocities and migration are optimized by cooperative L-selectin and intercellular adhesion molecule-1 functions.

Selectin family members largely mediate initial tethering and rolling of leukocytes on vascular endothelium, whereas integrin and Ig family members are essential for leukocyte firm adhesion. To quantify functional synergy between L-selectin and Ig family members during leukocyte rolling, the EA.hy926 human vascular endothelial line was transfected with either fucosyltransferase VII (926-FtVII) cDNA to generate L-selectin ligands alone or together with ICAM-1 cDNA (926-FtVII/ICAM-1). The ability of transfected 926 cells to support human leukocyte interactions was assessed in vitro using parallel plate flow chamber assays. Lymphocyte rolling on 926-FtVII cells was increased by approximately 70% when ICAM-1 was expressed at physiological levels. Although initial tether formation was similar for both cell types, lymphocyte rolling was 26% slower on 926-FtVII/ICAM-1 cells. Pretreatment of lymphocytes with an anti-CD18 mAb eliminated the increase in rolling, and all rolling was blocked by anti-L-selectin mAb. In addition, rolling velocities of lymphocytes from CD18-hypomorphic mice were 48% faster on 926-FtVII/ICAM-1 cells, with a similar reduction in rolling frequency relative to wild-type lymphocytes. CD18-hypomorphic lymphocytes also showed an approximately 40% decrease in migration to peripheral and mesenteric lymph nodes during in vivo migration assays compared with wild-type lymphocytes. Likewise, wild-type lymphocyte migration to peripheral lymph nodes was reduced by approximately 50% in ICAM-1(-/-) recipient mice. Similar to human lymphocytes, human neutrophils showed enhanced rolling interactions on 926-FtVII/ICAM-1 cells, but also firmly adhered. Thus, in addition to mediating leukocyte firm adhesion, CD18 integrin/ICAM-1 interactions regulate leukocyte rolling velocities and thereby optimize L-selectin-mediated leukocyte rolling.

A functional role for circulating mouse L-selectin in regulating leukocyte/endothelial cell interactions in vivo.

L-selectin mediates the initial capture and subsequent rolling of leukocytes along inflamed vascular endothelium and mediates lymphocyte migration to peripheral lymphoid tissues. Leukocyte activation induces rapid endoproteolytic cleavage of L-selectin from the cell surface, generating soluble L-selectin (sL-selectin). Because human sL-selectin retains ligand-binding activity in vitro, mouse sL-selectin and its in vivo relevance were characterized. Comparable with humans, sL-selectin was present in adult C57BL/6 mouse sera at approximately 1.7 micro g/ml. Similar levels of sL-selectin were present in sera from multiple mouse strains, despite their pronounced differences in cell surface L-selectin expression levels. Adhesion molecule-deficient mice prone to spontaneous chronic inflammation and mice suffering from leukemia/lymphoma had 2.5- and 20-fold increased serum sL-selectin levels, respectively. By contrast, serum sL-selectin levels were reduced by 70% in Rag-deficient mice lacking mature lymphocytes. The majority of serum sL-selectin had a molecular mass of 65-75 kDa, consistent with its lymphocyte origin. Slow turnover may explain the relatively high levels of sL-selectin in vivo. The t(1/2) of sL-selectin, assessed by transferring sera from wild-type mice into L-selectin-deficient mice and monitoring serum sL-selectin levels by ELISA, was >20 h, and it remained detectable for longer than 1 wk. Short-term in vivo lymphocyte migration assays demonstrated that near physiologic levels ( approximately 0.9 micro g/ml) of sL-selectin decreased lymphocyte migration to peripheral lymph nodes by >30%, with dose-dependent inhibition occurring with increasing sL-selectin concentrations. These results suggest that sL-selectin influences lymphocyte migration in vivo and that the increased sL-selectin levels present in certain pathologic conditions may adversely affect leukocyte migration.