Antony C. Bakke

Functional assay for human CD4+CD25+ Treg cells reveals an age-dependent loss of suppressive activity.

CD4+CD25+ regulatory T cells (Treg cells) prevent T cell‐mediated autoimmune diseases in rodents. To develop a functional Treg assay for human blood cells, we used FACS‐ or bead‐sorted CD4+CD25+ T cells from healthy donors to inhibit anti‐CD3/CD28 activation of CD4+CD25− indicator T cells. The data clearly demonstrated classical Treg suppression of CD4+CD25− indicator cells by both CD4+CD25+high and CD4+CD25+low T cells obtained by FACS or magnetic bead sorting. Suppressive activity was found in either CD45RO− (naive) or CD45RO+ (memory) subpopulations, was independent of the TCR signal strength, required cell–cell contact, and was reversible by interleukin‐2 (IL‐2). Of general interest is that a wider sampling of 27 healthy donors revealed an age‐ but not gender‐dependent loss of suppressive activity in the CD4+CD25+ population. The presence or absence of suppressive activity in CD4+CD25+ T cells from a given donor could be demonstrated consistently over time, and lack of suppression was not due to method of sorting, strength of signal, or sensitivity of indicator cells. Phenotypic markers did not differ on CD4+CD25+ T cells tested ex vivo from suppressive vs. nonsuppressive donors, although, upon activation in vitro, suppressive CD4+CD25+ T cells had significantly higher expression of both CTLA‐4 and GITR than CD4+CD25− T cells from the same donors. Moreover, antibody neutralization of CTLA‐4, GITR, IL‐10, or IL‐17 completely reversed Treg‐induced suppression. Our results are highly consistent with those reported for murine Treg cells and are the first to demonstrate that suppressive activity of human CD4+CD25+ T cells declines with age. Published 2003 Wiley‐Liss, Inc.

Direct enumeration and functional assessment of circulating dendritic cells in patients with liver disease.

Chronic liver disease has been shown to be associated with diminished humoral and cellular immune function. Although antigen‐presenting cells (APC) that initiate immune responses include various cells (B cells, endothelial cells, macrophages, etc.), the dendritic cell (DC) is a professional APC that activates naive T cells most efficiently. To examine the frequency and function of DCs in chronic liver disease, we studied circulating DCs from a cohort of 112 subjects (23 normal subjects, 29 subjects who had spontaneously recovered from hepatitis C virus [HCV] infection, 30 chronically infected HCV patients, and 30 patients with liver disease unrelated to HCV infection). Our analyses revealed significant reduction in both circulating myeloid (mDC) and plasmacytoid dendritic cells (pDC) in patients with liver disease. In contrast, examination of subjects with spontaneously resolved HCV infection revealed no significant difference in either circulating mDCs or pDCs. We found an inverse correlation with serum alanine aminotransferase (ALT) levels and both mDCs and pDCs frequency. In a subset of patients for whom intrahepatic cells were available, paired analysis revealed enrichment for DCs within the intrahepatic compartment. Interferon alfa (IFN‐α) production in response to influenza A and poly (I:C) correlated with the frequency of circulating DCs, although IFN‐α production was comparable on a per‐DC basis in patients with liver disease. In conclusion, patients with liver disease exhibit a reduction in circulating DCs. Considering that DCs are essential for initiation and regulation of innate and adaptive immunity, these findings have implications for both viral persistence and liver disease. (HEPATOLOGY 2004;40:335–345.)

Nucleosomes and DNA bind to specific cell-surface molecules on murine cells and induce cytokine production

The molecular basis for the cellular interaction of DNA and nucleosomes and the physiological consequences of this binding were examined. Both DNA and nucleosomes were demonstrated to bind specifically to the surface of human peripheral blood mononuclear cells and the murine T cell line S49. Western blots of S49 cell membranes, using probes of biotin-labeled DNA and nucleosomes, showed reactivity at 29 and 69 kDa. Functionally, the interaction of DNA and nucleosomes with murine spleen cells stimulated the release of significant amounts of IL-6 activity. There is evidence that nucleosomes, a product of apoptosis, are the major component of circulating DNA found in the plasma of patients with systemic lupus erythematosus (SLE). The interaction of nucleosomes with cell-surface DNA binding molecules may have physiological relevance to some of the immune aberrations observed in patients with SLE.

Alterations in T-Cell Subset Frequency in Peripheral Blood in Obesity

Background: Obesity affects the regulation of immune and inflammatory responses. This study characterizes differences in peripheral blood lymphocyte phenotype in obese humans. Methods: Frequencies of lymphocyte subsets among peripheral blood mononuclear cells were compared between 10 obese (BMI ≥35) and 10 lean subjects, as determined by antibodies directed against cluster differentiation (CD) markers. Results: Obese patients demonstrated an increased frequency of CD3+CD4+ T-cells (mean difference 12%, P=0.004), a decreased frequency of CD3+CD8+ T-cells (mean difference 9.4%, P=0.016) and an increased frequency of CD3+CD8+CD95+ T-cells (mean difference 13.3%, P=0.032). No other differences among T-cell or monocyte subsets were noted. Conclusions: Obesity is associated with alterations in frequencies of peripheral CD4+ and CD8+ T-cells and aberrations in the expression of CD95 among CD8+ T-cells. These data suggest both CD4+ and CD8+ T-cell compartments, as well as the regulation of CD95 expression on CD8+ T-cells, as targets for further study into obesitys effects on the immune system.

Neutrophil CD64 expression: distinguishing acute inflammatory autoimmune disease from systemic infections

Background: Common bacterial and opportunistic infections are a major cause of mortality in patients who are immunosuppressed owing to treatment with corticosteroids or cytotoxic drugs. Common laboratory tests for infection lack sensitivity and specificity. One of the new generation of tests to detect early systemic infections measures the up regulation of an Fc receptor (Fcγ R1, or CD64) on neutrophils. The Fc receptors on white blood cells are very important for effective phagocytosis of bacteria and are up regulated during an infection. Objective: To measure the clinical usefulness of quantitative CD64 measurements to differentiate between systemic infection and active autoimmune inflammation in an ongoing study. Methods: Patients with systemic infection (n=27), active autoimmune inflammatory disease (n=44), vasculitis (n=5), and controls (n=20) were studied for neutrophil CD64 expression using monoclonal antibodies and flow cytometry. Results: The median (interquartile range (IQR)) CD64 expression in patients with active inflammatory disease and systemic infection was 907.5 (586–1550) and 3647 (2380–6642), respectively (p<0.0001). The median (IQR) CD64 expression in control patients (osteoarthritis and fibromyalgia) was 505 (359–599). The sensitivity and specificity of CD64 expression on neutrophils to diagnose systemic infection (using a cut off value of 2000) was 85% and 91%, respectively. Conclusion: These results indicate that quantitative measurement of CD64 can distinguish between systemic infection and the flare of autoimmune diseases.

The effect of hypnotic-guided imagery on psychological well-being and immune function in patients with prior breast cancer.

OBJECTIVE To determine the effect of hypnotic-guided imagery on immune function and psychological parameters in patients being treated for Stage I or II breast cancer. METHODS To determine the effects of hypnotic-guided imagery on immune function and psychological parameters, the following study was undertaken. Psychological profiles, natural killer (NK) cell number and activity were measured at baseline, after the 8-week imagery training program and at the 3-month follow-up. RESULTS There were significant increases in improvement in depression (P<.04) and increase in absolute number of NK cells, but these were not maintained at the 3-month follow-up. Hypnotic-guided imagery did cause some transient changes in psychological well-being and immune parameters. However, these changes were not retained after the treatment ended. CONCLUSIONS Many studies during the last 15 years have demonstrated interactions between the central nervous and the immune systems. While a negative effect of stress on immune responses has been demonstrated, there have also been published reports that psychological treatments can positively alter the immune system. However, given the complexities of immune system kinetics, the transient nature of any psychological effect and the insensitivity of immune assays, our study indicates that there is a role for hypnotic-guided imagery as an adjuvant therapy.

Rescue of the autoimmune scurfy mouse by partial bone marrow transplantation or by injection with T-enriched splenocytes.

The scurfy mutant mouse is the genetic and phenotypic equivalent of the single‐gene human autoimmune disease immunodysregulation, polyendocrinopathy, enteropathy, X‐linked (IPEX). The scurfy mutation disrupts the Foxp3 gene, a putative master switch for T regulatory cell development. Bone marrow transplant without conditioning was previously reported to be ineffective in scurfy mice, yet clinical remission occurs in transplanted human IPEX patients despite limited donor engraftment. In view of this contradiction, we sought to validate scurfy as a model for studying the pathogenesis and treatment of human IPEX, in particular the phenomenon of dominant immune regulation. One half of scurfy mice given bone marrow transplants after sublethal irradiation recovered and survived long‐term with donor chimerism ranging from 1·7% to 50%. Early transfer of 2 × 107 normal T cell‐enriched splenocytes also prevented or limited disease and permitted long‐term survival. Donor T cells in rescued mice made up 3–5% of lymphocytes and became highly enriched for CD25+ T cells over time. Transfer of 106 CD4+ CD25+ sorted T cells showed some beneficial effect, while CD4+ CD25‐ cells did not. Thus, both partial bone marrow transplant and T‐enriched splenocyte transfer are effective treatments for scurfy. These results indicate that scurfy results from a lack of cells with dominant immune regulatory capacity, possibly T regulatory cells. The potency of small numbers of normal cells indicates that IPEX may be a feasible target for gene therapy.

Dynamics of Neisseria gonorrhoeae Attachment: Microcolony Development, Cortical Plaque Formation, and Cytoprotection

ABSTRACT Neisseria gonorrhoeae is the bacterium that causes gonorrhea, a major sexually transmitted disease and a significant cofactor for human immunodeficiency virus transmission. The retactile N. gonorrhoeae type IV pilus (Tfp) mediates twitching motility and attachment. Using live-cell microscopy, we reveal for the first time the dynamics of twitching motility by N. gonorrhoeae in its natural environment, human epithelial cells. Bacteria aggregate into microcolonies on the cell surface and induce a massive remodeling of the microvillus architecture. Surprisingly, the microcolonies are motile, and they fuse to form progressively larger structures that undergo rapid reorganization, suggesting that bacteria communicate with each other during infection. As reported, actin plaques form beneath microcolonies. Here, we show that cortical plaques comigrate with motile microcolonies. These activities are dependent on pilT, the Tfp retraction locus. Cultures infected with a pilT mutant have significantly higher numbers of apoptotic cells than cultures infected with the wild-type strain. Inducing pilT expression with isopropyl-β-d-thiogalactopyranoside partially rescues cells from infection-induced apoptosis, demonstrating that Tfp retraction is intrinsically cytoprotective for the host. Tfp-mediated attachment is therefore a continuum of microcolony motility and force stimulation of host cell signaling, leading to a cytoprotective effect.

T-cell receptor variable β genes show differential expression in CD4 and CD8 T cells

Abstract Studies in transgenic and inbred strains of mice have shown that the critical molecular interactions controlling positive selection involve major histocompatibility complex (MHC), T-cell receptor (TCR), and CD4 or CD8 coreceptor molecules. Correlations have been established between MHC gene products and the percentage of CD4 or CD8 T cells that express specific variable (V) β-gene products as part of the αβ heterodimer. These studies have important implications regarding potential mechanisms of HLA-linked autoimmune diseases in humans. If similar interactions are required for positive selection in humans, one would predict that the TCR repertoire expressed by mature, peripheral blood CD4 and CD8 T cells would vary. To test this hypothesis the expression of specific TCR Vβ-region genes by CD4 and CD8 T cells from healthy individuals was compared using both triple-color flow cytometry and polymerase chain reaction based experimental approaches. The results show that the TCR repertoire does vary as a function of CD4 and CD8 T-cell subsets. Among unrelated individuals certain Vβ genes were consistently overrepresented in the CD4 population (Vβ-5.1, −6.7a, and −18); some were skewed to the CD8 population (Vβ-14) while others showed variable patterns (Vβ-12 and −17). Deletion of entire Vβ gene families was not observed suggesting that this is a rare event in humans. Attempts to correlate the expressed TCR repertoire in humans with HLA alleles will require consideration of these differences in expression as a function of subset.

Estrogen treatment induces a novel population of regulatory cells, which suppresses experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a debilitating neurological disease characterized by a progressive loss of motor and sensory function, eventually leading to paralysis and death. The primary cause of neurological impairment is demyelination of the central nervous system (CNS) caused by an inflammatory autoimmune response. Previous studies have shown that the severity of MS is reduced during pregnancy, suggesting that the increased level of sex hormones may reduce the autoimmune response. Recently, we have shown that estrogen treatment confers protection from experimental autoimmune encephalomyelitis (EAE), which is an animal model for MS. However, the cellular basis of estrogens action remains unknown. In the current study, we demonstrate that estrogen treatment led to the induction of a novel subpopulation of regulatory cells in spleen and CNS, which also occurs naturally in pregnant mice. These previously uncharacterized cells display a low level expression of CD45 (CD45dim) and no detectable expression of many cell surface markers related to TCR signaling, including CD3 and TCR. However, these cells retained expression of VLA‐4, an extracellular protein involved in cellular migration. Several lines of evidence suggest that these novel cells, defined as CD45dimVLA‐4+ cells, may play a role in the protective effects of estrogen in EAE. Injection of purified CD45dimVLA‐4+ cells conferred protection from spontaneous EAE (Sp‐EAE). In contrast, injection of CD45highVLA‐4+ cells exacerbated the disease course. CD45dimVLA‐4+ cells also suppressed antigen‐specific proliferation of primed lymphocytes in coculture. A better understanding of how CD45dimVLA‐4+ cells suppress the harmful immune response of EAE may help in explaining the induction of immune tolerance during pregnancy and lead to novel therapeutic approaches to combat MS and other autoimmune diseases.