The measurement of whole blood (WB) concentrations has been the primary method for therapeutic drug monitoring of tacrolimus since its introduction in the field of organ transplantation. However, ... Show moreThe measurement of whole blood (WB) concentrations has been the primary method for therapeutic drug monitoring of tacrolimus since its introduction in the field of organ transplantation. However, >99% of tacrolimus measured in WB is bound to erythrocytes and plasma proteins, which are the pharmacologically inactive fractions. The pharmacologically active fractions, the free (or unbound) tacrolimus in plasma and the intracellular tacrolimus, make up 1% or less of the WB concentration. The mechanism of action of tacrolimus is to inhibit the enzyme calcineurin within T lymphocytes and, therefore, measuring the intralymphocytic tacrolimus concentration may better reflect its pharmacodynamic effects and better correlate with clinical outcomes. However, studies on intracellular tacrolimus concentrations have shown conflicting results. In this review, we argue that we need to overcome the analytical limitations of current assays for the measurement of intracellular tacrolimus before moving this technique into the clinical setting. The validity and standardization of the cell isolation process before the measurement of the intracellular tacrolimus concentration is as important as the measurement itself but has received little attention in our view. Recent evidence suggests that the addition of an inhibitor of P-glycoprotein, an efflux transporter expressed on lymphocytes, prevents the expulsion of tacrolimus during the cell isolation process. Refining the technique for the intracellular tacrolimus concentration measurement should be the focus followed by clinical evaluation of its association with rejection risk. Show less
Long-lived parasites evade host immunity through highly evolved molecular strategies. The murine intestinal helminth, Heligmosomoides polygyrus, down-modulates the host immune system through... Show moreLong-lived parasites evade host immunity through highly evolved molecular strategies. The murine intestinal helminth, Heligmosomoides polygyrus, down-modulates the host immune system through release of an immunosuppressive TGF-β mimic, TGM1, which is a divergent member of the CCP (Sushi) protein family. TGM1 comprises 5 domains, of which domains 1-3 (D1/2/3) bind mammalian TGF-β receptors, acting on T cells to induce Foxp3+ regulatory T cells; however, the roles of domains 4 and 5 (D4/5) remain unknown. We noted that truncated TGM1, lacking D4/5, showed reduced potency. Combination of D1/2/3 and D4/5 as separate proteins did not alter potency, suggesting that a physical linkage is required and that these domains do not deliver an independent signal. Coprecipitation from cells treated with biotinylated D4/5, followed by mass spectrometry, identified the cell surface protein CD44 as a coreceptor for TGM1. Both full-length and D4/5 bound strongly to a range of primary cells and cell lines, to a greater degree than D1/2/3 alone, although some cell lines did not respond to TGM1. Ectopic expression of CD44 in nonresponding cells conferred responsiveness, while genetic depletion of CD44 abolished enhancement by D4/5 and ablated the ability of full-length TGM1 to bind to cell surfaces. Moreover, CD44-deficient T cells showed attenuated induction of Foxp3 by full-length TGM1, to levels similar to those induced by D1/2/3. Hence, a parasite protein known to bind two host cytokine receptor subunits has evolved a third receptor specificity, which serves to raise the avidity and cell type–specific potency of TGF-β signaling in mammalian cells. Show less
Objective: To determine the effect of tetanus toxoid (TT) revaccination on circulating B-, T-and NK-cell com-partments in myasthenia gravis (MG) patients.Methods: Lymphocyte (sub)populations and... Show moreObjective: To determine the effect of tetanus toxoid (TT) revaccination on circulating B-, T-and NK-cell com-partments in myasthenia gravis (MG) patients.Methods: Lymphocyte (sub)populations and differentiation stages were assessed by flow cytometry in 50 TT revaccinated MG patients. TT-specific proliferative responses were explored in PBMC cultures. Results: In patients treated with azathioprine B-and NK cell numbers were strongly decreased. Lymphocyte (sub) populations remained unaffected upon TT revaccination. t All patients showed a significant TT-induced prolif-erative response.Conclusion: TT revaccination is effective in MG patients with stable disease irrespective of their thymectomy status and medication and does not alter the composition of the lymphocyte compartment. Show less
T cell responses upon infection display a remarkably reproducible pattern of expansion, contraction, and memory formation. If the robustness of this pattern builds entirely on signals derived from... Show moreT cell responses upon infection display a remarkably reproducible pattern of expansion, contraction, and memory formation. If the robustness of this pattern builds entirely on signals derived from other cell types or if activated T cells themselves contribute to the orchestration of these population dynamics-akin to bacterial quorum regulation-is unclear. Here, we examined this question using time-lapse microscopy, genetic perturbation, bioinformatic predictions, and mathematical modeling. We found that ICAM-1-mediated cell clustering enabled CD8(+) T cells to collectively regulate the balance between proliferation and apoptosis. Mechanistically, T cell expressed CD80 and CD86 interacted with the receptors CD28 and CTLA-4 on neighboring T cells; these interactions fed two nested antagonistic feedback circuits that regulated interleukin 2 production in a manner dependent on T cell density as confirmed by in vivo modulation of this network. Thus, CD8(+) T cell-population-intrinsic mechanisms regulate cellular behavior, thereby promoting robustness of population dynamics. Show less
The immune system of mammals is responsible for protecting our body against pathogensand foreign substances (antigens), and it consists of two discrete lines of defense. The firstline called innate... Show moreThe immune system of mammals is responsible for protecting our body against pathogensand foreign substances (antigens), and it consists of two discrete lines of defense. The firstline called innate immunity and provide a quick and nonspecific defense. The innate immunityincludes different cells types, such as mast cells, macrophages, neutrophils, eosinophils,dendritic cells and natural killer (NK) cells. The second line of defense called adaptive immunityresponds in an antigen-specific manner, and comprised of B and T lymphocyte cells.This thesis focuses on one of the signals which are known to play an important role duringHSC repopulation and T cell development that is Wnt signaling pathway. Dependingon the tissue/cell types (microenvironment) and specific class of Wnt proteins binding tothe corresponding receptors on the developing lymphocytes, two discrete downstreampathways will be activated namely canonical or non-canonical Wnt pathway. The main aimof this thesis is to dissect the roles of these two distinct pathways during hematopoiesisand lymphocyte development in murine as a physiologically relevant animal model. Show less
Following antigen encounter, activated T cells can give rise to functionally distinct T cell subsets. Understanding how different T cell subsets arise requires technologies that can monitor the... Show moreFollowing antigen encounter, activated T cells can give rise to functionally distinct T cell subsets. Understanding how different T cell subsets arise requires technologies that can monitor the developmental potential of single precursor cells (chapter 2). This thesis describes the development and use of two novel genetic tagging strategies aimed at following cell differentiation in vivo. These strategies are based on the marking of precursor cells with unique DNA sequences (barcodes), following which cell fate is analyzed by barcode comparison of different daughter populations. The first technology, termed cellular barcoding, makes use of a retroviral barcode library to provide T cells with unique genetic tags via in vitro transduction. Cellular barcoding was used to analyze the kinship of diverse T cell populations (chapter 3-5) as well as to measure the clonality of antigen-specific T cell responses under varying conditions of infection (chapter 6). The second technology, termed in vivo barcoding, makes use of a transgenic mouse model in which unique DNA sequences are introduced via inducible VDJ recombination. The feasibility of in vivo barcoding was demonstrated by conditionally labeling lung and liver cells with different barcodes (chapter 7). Together, these studies have yielded important new insights for vaccine optimization. Show less
