Karen M. Smith

In situ characterization of CD4 T cell behavior in mucosal and systemic lymphoid tissues during the induction of oral priming and tolerance

The behavior of antigen-specific CD4+ T lymphocytes during initial exposure to antigen probably influences their decision to become primed or tolerized, but this has not been examined directly in vivo. We have therefore tracked such cells in real time, in situ during the induction of oral priming versus oral tolerance. There were marked contrasts with respect to rate and type of movement and clustering between naive T cells and those exposed to antigen in immunogenic or tolerogenic forms. However, the major difference when comparing tolerized and primed T cells was that the latter formed larger and longer-lived clusters within mucosal and peripheral lymph nodes. This is the first comparison of the behavior of antigen-specific CD4+ T cells in situ in mucosal and systemic lymphoid tissues during the induction of priming versus tolerance in a physiologically relevant model in vivo.

Mesenteric lymph nodes at the center of immune anatomy

The surface of the intestinal mucosa is constantly assaulted by food antigens and enormous numbers of commensal microbes and their products, which are sampled by dendritic cells (DCs). Recent work shows that the mesenteric lymph nodes (MLNs) are the key site for tolerance induction to food proteins and that they also act as a firewall to prevent live commensal intestinal bacteria from penetrating the systemic immune system.

Efficacy of common hospital biocides with biofilms of multi-drug resistant clinical isolates

The hospital environment is particularly susceptible to contamination by bacterial pathogens that grow on surfaces in biofilms. The effects of hospital biocides on two nosocomial pathogens, meticillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, growing as free-floating (planktonic) and adherent biofilm populations (sessile) were examined. Clinical isolates of MRSA and P. aeruginosa were grown as biofilms on discs of materials found in the hospital environment (stainless steel, glass, polyethylene and Teflon) and treated with three commonly used hospital biocides containing benzalkonium chloride (1 % w/v), chlorhexidine gluconate (4 % w/v) and triclosan (1 % w/v). Cell viability following biocide treatment was determined using an XTT assay and the LIVE/DEAD BacLight Bacterial Viability kit. The minimum bactericidal concentration (MBC) of all biocides for planktonic populations of both organisms was considerably less than the concentration recommended for use by the manufacturer. However, when isolates were grown as biofilms, the biocides were ineffective at killing bacteria at the concentrations recommended for use. Following biocide treatment, 0-11 % of cells in MRSA biofilms survived, and up to 80 % of cells in P. aeruginosa biofilms survived. This study suggests that although biocides may be effective against planktonic populations of bacteria, some biocides currently used in hospitals are ineffective against nosocomial pathogens growing as biofilms attached to surfaces and fail to control this reservoir for hospital-acquired infection.

Th1 and Th2 CD4+ T cells provide help for B cell clonal expansion and antibody synthesis in a similar manner in vivo.

The relative ability of Th1 and Th2 T cells to help B cells remains controversial as do the mechanisms by which both T cell subsets provide help in vivo. Whether this help affects the clonal expansion and/or differentiation of B cells has been difficult to assess due to the low frequency of Ag-specific T and B lymphocytes. We have employed a novel technique to directly monitor the clonal expansion of Ag-specific T and B lymphocytes in vivo. OVA-specific TCR transgenic T lymphocytes were polarized toward a Th1 or Th2 phenotype in vitro. These cells were then transferred into syngeneic recipients, along with B cell receptor transgenic hen egg lysozyme-specific B lymphocytes. Our results indicate that Th1 and Th2 cells support B cell responses to a similar extent in vivo and that they achieve this in the same manner by migrating into B cell follicles to promote CD154-dependent B cell clonal expansion and Ab production.

The Anatomy of Mucosal Immune Responses

Abstract: It remains unclear how and where unresponsiveness to fed antigens is induced. This “oral tolerance” is probably necessary to prevent the array of immune effector mechanisms required to counteract pathogens of the mucosae from being misdirected against food antigens or commensal flora. It will obviously be important to dissect where, when, and how such immunological homoestasis is maintained in the gut, but it will also be necessary to determine whether similar inductive and effector mechanisms are required for the therapeutic applications of oral tolerance systemically. This may be influenced by anatomical and microenvironmental effects on the phenotype and/or activation state of the antigen‐presenting cell (APC), which presents orally delivered antigen. Fed antigen passes from the intestinal lumen either via the villus epithelium and M cells in the Peyers patches (PP) or the mucosal lamina propria to the organized lymphoid tissues of the PP and mesenteric lymph nodes (MLN). In addition, there is evidence that mucosally administered antigen also gains access directly to peripheral lymphoid organs. Each of these sites contains distinctive populations of APCs and has unique local microenvironments that may influence the immune response in different ways. We propose that feeding antigen in high doses may induce clonal anergy, deletion, or altered differentiation because it gains direct access to resting APCs in the T cell areas of both the gut‐associated lymphoid tissues (GALT) and peripheral lymphoid organs, with presentation occurring in the absence of productive costimulation. By contrast, low doses of tolerizing antigen may be taken up and presented preferentially by APCs in the GALT, where the local environment may favor the induction of regulatory T cells. This is consistent with our own and others findings, using adoptive transfer of TcR tg T cells. These studies have shown that antigen‐specific CD4+ T cells are activated simultaneously in all peripheral and gut‐associated lymphoid organs after feeding high doses of proteins, but that this may be more restricted to local tissues when lower doses are used. Another level of anatomical control is imposed within lymphoid organs, where migration of T cells through distinct anatomical compartments can affect their differentiation. We find that, in contrast to orally primed T cells, orally tolerized T cells are unable to migrate into B cell follicles during their initial exposure to antigen. This affects their differentiation as upon subsequent challenge with antigen in adjuvant, tolerized T cells can be found in follicles but are unable to provide the B cell help that primed T cells can deliver. We hypothesize that the initial defective migration of tolerized T cells prevents them from receiving signals from antigen‐specific B cells in follicles and results in abortive differentiation. Thus, both gross and fine anatomical location of fed antigen presentation may be important in mucosal immunoregulation.

T-cell activation occurs simultaneously in local and peripheral lymphoid tissue following oral administration of a range of doses of immunogenic or tolerogenic antigen although tolerized T cells display a defect in cell division

How the mucosal immune system promotes active immunity against harmful organisms but tolerance to commensal bacteria or dietary antigens is poorly understood. Thus, the antigen‐presenting cell (APC), site of antigen presentation, and effector mechanisms responsible for oral priming and tolerance remain unclear. Characterizing differences between oral priming and tolerance may improve the exploitation of oral tolerance for therapeutic applications and aid the design of oral vaccines. To address these questions we compared the mucosal and systemic activation and localization of antigen‐specific T cells during the induction of oral priming and tolerance. Activation marker expression and cell division by tg T cells was determined in conjunction with their anatomical location. These studies show that after feeding, T cells are activated in both peripheral and local lymphoid tissues within 6 hr, irrespective of the presence of adjuvant. Subsequently, T‐cell accumulation can be detected simultaneously in peripheral and mesenteric lymph nodes and Peyers patches within 24 hr of feeding, but only after 3 days post feeding in the lamina propria. Primed and tolerized T cells adopted similar phenotypes as assessed by activation marker expression. However, within the mesenteric lymph nodes (MLN) tolerized T cells underwent significantly fewer divisions than primed T cells. Thus, T‐cell activation and expansion occurs throughout the animal after feeding a range of doses of antigen, irrespective of whether priming or tolerance is the eventual outcome. However, the presence of an adjuvant enhances clonal expansion in the MLN while tolerized T cells display defective cell division.

Inducible Costimulatory Molecule-B7-Related Protein 1 Interactions Are Important for the Clonal Expansion and B Cell Helper Functions of Naive, Th1, and Th2 T Cells

Inducing T cell responses requires at least two distinct signals: 1) TCR engagement of MHC-peptide and 2) binding of CD28 to B7.1/2. However, the recent avalanche of newly described costimulatory molecules may represent additional signals which can modify events after the initial two-signal activation. Inducible costimulatory molecule (ICOS) is a CD28 family member expressed on T cells rapidly following activation that augments both Th1 and Th2 T cell responses and has been implicated in sustaining rather than initiating T cell responses. Although it is known that blockade of ICOS-B7-related protein 1 (B7RP-1) in vivo dramatically reduces germinal center formation and Ab production, the mechanism(s) remains unclear. An optimal T cell-dependent Ab response requires T and B cell activation, expansion, differentiation, survival, and migration, and the ICOS-B7RP-1 interaction could be involved in any or all of these processes. Understanding this will have important implications for targeting ICOS-B7RP-1 therapeutically. We have therefore used a double-adoptive transfer system, in which all of the above events can be analyzed, to assess the role of ICOS-B7RP-1 in T cell help for B cell responses. We have shown that ICOS signaling is involved in the initial clonal expansion of primary and primed Th1 and Th2 cells in response to immunization. Furthermore, while ICOS-B7RP-1 interactions have no effect on the migration of T cells into B cell follicles, it is essential for their ability to support B cell responses.

In Vivo Generated Th1 Cells Can Migrate to B Cell Follicles to Support B Cell Responses

The description of Th1 and Th2 T cell subsets rationalized the inverse correlation between humoral and cell-mediated immunity. Although Th1 cells were described to support cell-mediated immune responses, their role in supporting certain B cell responses was firmly established. However, there is now a prevailing preconception that provision of B cell help is entirely the domain of Th2 cells and that Th1 cells lack this capacity. Previous studies demonstrated that immunization using aluminum hydroxide adjuvants induces Ag-specific Th2 responses, whereas incorporation of IL-12 with aluminum hydroxide produces a Th1 inducing adjuvant. By immunizing TCR transgenic recipient mice in this fashion, we have generated Ag-specific, traceable Th1 and Th2 cells in vivo and assessed their follicular migration and ability to support B cell responses. In this study we have shown that in vivo polarized Th1 and Th2 cells clonally expand to similar levels and migrate into B cell follicles in which they support B cell responses to a similar degree. Critically, we present direct evidence that in vivo polarized, IFN-γ secreting Th1 cells migrate into B cell follicles where they can interact with Ag-specific B cells.

Influence of Tigecycline on Expression of Virulence Factors in Biofilm-Associated Cells of Methicillin-Resistant Staphylococcus aureus

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) infections are complicated by the ability of the organism to grow in surface-adhered biofilms on a multitude of abiotic and biological surfaces. These multicellular communities are notoriously difficult to eradicate with antimicrobial therapy. Cells within the biofilm may be exposed to a sublethal concentration of the antimicrobial due to the metabolic and phenotypic diversity of the biofilm-associated cells or the protection offered by the biofilm structure. In the present study, the influence of a sublethal concentration of tigecycline on biofilms formed by an epidemic MRSA-16 isolate was investigated by transcriptome analysis. In the presence of the drug, 309 genes were upregulated and 213 genes were downregulated by more than twofold in comparison to the levels of gene regulation detected for the controls not grown in the presence of the drug. Microarray data were validated by real-time reverse transcription-PCR and phenotypic assays. Tigecycline altered the expression of a number of genes encoding proteins considered to be crucial for the virulence of S. aureus. These included the reduced expression of icaC, which is involved in polysaccharide intercellular adhesin production and biofilm development; the upregulation of fnbA, clfB, and cna, which encode adhesins which attach to human proteins; and the downregulation of the cap genes, which mediate the synthesis of the capsule polysaccharide. The expression of tst, which encodes toxic shock syndrome toxin 1 (TSST-1), was also significantly reduced; and an assay performed to quantify TSST-1 showed that the level of toxin production by cells treated with tigecycline decreased by 10-fold (P < 0.001) compared to the level of production by untreated control cells. This study suggests that tigecycline may reduce the expression of important virulence factors in S. aureus and supports further investigation to determine whether it could be a useful adjunct to therapy for the treatment of biofilm-mediated infections.

Orally tolerized T cells are only able to enter B cell follicles following challenge with antigen in adjuvant, but they remain unable to provide B cell help.

Although it is well documented that feeding Ag can tolerize or prime systemic humoral and cell-mediated immune responses, the mechanisms involved remain unclear. Elucidation of these mechanisms remains, in part, complicated by the inability to assess responses by individual lymphocyte populations. In the past, in vivo studies have examined T cell responses at the gross level by examining their ability to support B cell Ab production. However, as the fed Ag has the capacity to affect B cells directly, analyzing the functional capacity of a single Ag-specific T cell population in vivo has been difficult. Using a double-adoptive transfer system, we have primed or tolerized T cells, independently of B cells with a high dose of fed Ag, and examined the ability of these primed or tolerized T cells to support B cell clonal expansion in response to a conjugated Ag in vivo. We have been able to show that primed T cells support B cell clonal expansion and Ab production whereas tolerized T cells do not. Thus, we have provided direct evidence that tolerized T cells are functionally unable to help B cells in vivo. Furthermore, we have shown that this inability of tolerized T cells to support fulminant B cell responses is not a result of defective clonal expansion or follicular migration, since following challenge tolerized T cells are similar to primed T cells in both of these functions.