Melodie-Jo Wolfs

Publications (3)13.09 Total impact

• Article: TRP expression pattern and the functional importance of TRPC3 in primary human T-cells.

  Anna S Wenning, Katherina Neblung, Bettina Strauss, Melodie-Jo Wolfs, Anne Sappok, Markus Hoth, Eva C Schwarz
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  ABSTRACT: TRP proteins form ion channels which are activated following receptor stimulation. In T-cell lines, expression data of TRP proteins have been published. However, almost no data about TRP expression is available in primary human T-cells. Using RT-PCR and quantitative RT-PCR, we compare the expression of TRP mRNA in 1) human peripheral blood lymphocytes, which are a mix of mostly mono-nuclear blood lymphocytes but contain other leucocytes, 2) a pure human CD4+ T-helper cell population in the resting (=naïve) and activated (=effector) state, and 3) two commonly used CD4+ Jurkat T-cell lines, E6-1 and parental. To mimic physiological cell stimulation, we analyzed TRP expression in primary human cells in a quantitative way over several days following formation of an immunological synapse through stimulation with antibody-coated beads. The TRP expression profile of primary human T-cells was significantly different from Jurkat T-cells. Among the TRP mRNAs of the TRPC, TRPM, and TRPV family, we found consistent expression of TRPC1, TRPC3, TRPV1, TRPM2, and TRPM7 in primary human CD4+ T-cells of all analyzed blood donors. Among these, TRPC3 and TRPM2 were strongly up-regulated following stimulation, but with different kinetics. We found that TRPC3 modulates Ca²+-dependent proliferation of primary CD4+ T-cells indicating that TRPC3 may be involved in Ca²+ homeostasis in T-cells besides the well-established STIM and ORAI proteins which are responsible for store-operated Ca²+ entry.
  Biochimica et Biophysica Acta 01/2011; 1813(3):412-23. · 4.66 Impact Factor
• Article: Efficiency of T-cell costimulation by CD80 and CD86 cross-linking correlates with calcium entry.

  Markus Thiel, Melodie-Jo Wolfs, Stefan Bauer, Anna S Wenning, Tanja Burckhart, Eva C Schwarz, Andrew M Scott, Christoph Renner, Markus Hoth
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  ABSTRACT: Costimulation is a fundamental principle of T-cell activation. In addition to T-cell receptor engagement, the interaction between CD80 and/or CD86 with CD28 and/or cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptors is required to regulate T-cell activation and tolerance. While the importance of costimulation is clearly established, the exact molecular mechanism is unknown. We demonstrate that T-cell proliferation and the ability of CD8(+) T-effector cells to kill were enhanced slightly by CD80 but dramatically by CD86 costimulation. To further analyse the cellular process of costimulation, we developed a single-cell assay to analyse Ca(2+) signals following costimulation with bi-specific antibodies. We found that this stimulation method worked in every human T-cell that was analysed, making it one of the most efficient T-cell activation methods to date for primary human T cells. The enhanced proliferation and killing by costimulation was paralleled by an increase of Ca(2+) influx following CD86 costimulation and it was dependent on CD28/CTLA-4 expression. The enhanced Ca(2+) influx following CD86 costimulation was abrogated by an antibody that interfered with CD28 function. The differences in Ca(2+) influx between CD80 and CD86 costimulation were not dependent on the depletion of Ca(2+) stores but were eliminated by the application of 10 mum 2-aminoethyldiphenyl borate which has recently been shown to enhance stromal interaction molecule 2 (STIM2)-dependent Ca(2+) entry while reducing STIM1-dependent Ca(2+) entry. Our data indicate that differences in the efficiency of costimulation are linked to differences in Ca(2+) entry.
  Immunology 10/2009; 129(1):28-40. · 3.32 Impact Factor
• Article: Calcium dependence of T cell proliferation following focal stimulation.

  Eva C Schwarz, Carsten Kummerow, Anna S Wenning, Kerstin Wagner, Anne Sappok, Katherina Waggershauser, Désirée Griesemer, Bettina Strauss, Melodie-Jo Wolfs, Ariel Quintana, Markus Hoth
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  ABSTRACT: Clonal T cell expansion through proliferation is a central process of the adaptive immune response. Apoptosis of activated T cells is required to avoid chronic inflammation. T cell proliferation and apoptosis are often analyzed with stimuli that do not induce formation of a functional immunological synapse. Here we analyze the Ca(2+) dependence of proliferation and apoptosis in primary human CD4(+) T cells following stimulation with anti-CD3/anti-CD28-coated beads, which induce a tight interaction similar to the immunological synapse. We found this focal stimulation to be much more efficient for stimulating IL-2 production and proliferation than non-focal TCR stimuli. Surprising little Ca(2+) entry through Ca(2+) channels was required for T cell proliferation. Transient free intracellular calcium concentration ([Ca(2+)](i)) elevations of up to 220 nM from a baseline level of around 40 nM were sufficient for maximal proliferation in primary human CD4(+) T cells. We also show that proliferation was very Ca(2+) sensitive in the range 90-120 nM, whereas apoptosis was basically constant for [Ca(2+)](i) levels of 90-120 nM. We conclude that very small changes in [Ca(2+)](i) can dramatically change the ratio between proliferation and apoptosis, thus keeping the balance between overshooting and inefficient immune responses.
  European Journal of Immunology 11/2007; 37(10):2723-33. · 5.10 Impact Factor