Oxidative signaling in T cells

ROS in TCR signaling
© modified from Kaminski et al., 2007, Mol Cell Biol 27, 3625-3639

Reactive oxygen species (ROS) play a key role in regulation of induction and termination of a T cell immune response by controlling gene expression (Gülow et al. 2005, summarized in Gülow et al. 2006 and Krammer et al. 2007). The molecular source and the signaling steps necessary for ROS production were largely unknown. Recently, we have shown that the proximal T-cell receptor (TCR)-signaling machinery, including protein kinase C θ (PKCθ),is crucial for ROS production. PKCθ translocates to the mitochondria upon stimulation. We identified the mitochondria as the source of activation-induced ROS. Here it is complex I of the respiratory chain that releases ROS (Kaminski et al. 2007). These ROS activate redox sensitive transcription factors (e.g. NF-κB and AP-1) and thereby induce TCR-dependent gene expression (e.g. IL-2, IL-4 and CD95L) (Kaminski et al. 2007 and Kaminski et al. 2010).

HIV-Tat and sensitization towards activation-induced T cell death (AICD)

In AIDS patients, AICD is strongly enhanced and accelerated. We and others have previously shown that HIV-1 trans-activator of transcription (HIV-Tat) sensitizes T cells towards CD95-mediated apoptosis by upregulating CD95L expression. We have demonstrated that HIV-Tat interferes with TCR signaling by induction of an oxidative signal. ROS generated by HIV-Tat combine with CD4-dependent calcium influx and cause massive T cell apoptosis via upregulation of CD95L. Thus, our data provide an explanation for CD4+ T lymphocyte depletion during progression of AIDS (Gülow et al. 2005).

Oxidative stress induces lymphoma cell death

Aberrant signaling of the NF-κB pathway has been identified as a mediator of survival and apoptosis resistance in leukemia and lymphoma. We have shown that inhibition of the constitutive active NF-κB pathway in cutaneous T cell lymphoma (CTCL) induces a down-regulation of ferritin heavy chain (FHC). This leads to an increase of free intracellular iron which in turn induces massive generation of ROS and cell death. In addition, in a murine T cell lymphoma model, we have demonstrated that inhibition of NF-κB and subsequent down-regulation of FHC significantly delays tumor growth in vivo. Thus, our results promote FHC as a potential target for effective therapy of lymphoma with aberrant NF-κB signaling (Klemke et al. 2009, Kiessling et al. 2009 and Kiessling et al. 2010).

KRAS and NRAS mutations sensitizing tumors towards inhibition of the RAS/RAF/MEK signaling cascade

We screened 90 biopsy specimens from CTCL patients for somatic mutations using OncoMap technology. We detected oncogenic mutations for the RAS pathway in 4/90 samples. All mutations were found in stage IV patients who showed significantly decreased overall survival compared to stage IV patients without mutations. In addition, we detected a RAS mutation in the CTCL cell line Hut78. Knock-down of NRAS by siRNA induced apoptosis in mutant Hut78 cells but not in CTCL cell lines lacking RAS mutations. The RAS mutation sensitized Hut78 cells towards growth inhibition by MEK inhibitors. Furthermore, we found that MEK inhibitors exclusively induce apoptosis in Hut78 cells. In summary, we conclude that RAS mutations are rare events at a late stage of CTCL and our preclinical results suggest that such late stage patients will profit from treatment with MEK inhibitors (Kiessling and Oberholzer et al. 2011).

Aging of the immune system

Multicellular organisms undergo qualitative changes during aging that are associated with progressive degeneration of biological functions, increased susceptibility to diseases, and increased probability of death within a given time period. Genetic evidence linking the generation of ROS to life span has been obtained for different animal species.Recently, we could show that one particular protein, which we have named Aging-Factor-1 (AF-1), is a critical negative regulator of the anti-oxidative defense system. AF-1 was dramatically upregulated in primary T cells from aged (>55 years) donors compared to T cells that were isolated from young donors (20-25 years). This finding applies to several other human cell types (e.g. hepatocytes, monocytes, fibroblasts). The increased AF-1 expression interferes with oxidative stress resistance. Therefore, we assume that alterations in ROS generation and destabilization of the redox equilibrium contribute to age-related immune dysfunction and inflammatory processes. We suggest that AF-1 plays a decisive role in the aging immune system by deregulating ROS-induced gene expression. Moreover, knocking down AF-1 in Drosophila melanogaster substantially enhances its life span. Our AF-1 knockdown flies appear more active, show an increased stress resistance, higher fertility and are long-lived. Indeed these flies age without prominent signs of ailing. Thus, aging is a phenomenon directly confined to single cells. Summation of aging of single cells results in aging of the organism. We propose AF-1 as a general regulator of aging and suggest that these exciting data provide a deeper insight into the ROS mediated aging process.

Key References

  1. Kaminski, M., Kiessling, M., Suss, D., Krammer, P. H., and Gülow, K. (2007). Novel role for mitochondria: protein kinase Ctheta-dependent oxidative signaling organelles in activation-induced T-cell death. Mol Cell Biol 27, 3625-3639.
  2. Kiessling, M., Klemke, C.-D., Kaminski, M., Galani, I. E., Krammer, P. H., and Gülow, K. (2009). Inhibition of constitutively activated nuclear factor-kappaB induces reactive oxygen species- and iron-dependent cell death in cutaneous T-cell lymphoma. Cancer Res 6, 2365-2374.
  3. Klemke, C.-D., Brenner D., Weiss E.-M., Schmidt M., Leverkus M., Gülow K., Peter H. Krammer. (2009) Lack of T cell receptor-stimulated CD95 ligand up-regulation protects cutaneous T cell lymphoma cells from Activation-Induced Cell Death (AICD). Cancer Res 10, 4175-4183.
  4. Kaminski M.M., Sauer S.W., Klemke C.-D., Süss D, Okun J.G., Krammer P.H., and Gülow K. (2010). Mitochondrial ROS control T cell activation by regulating IL-2 and IL-4 expression: mechanism of ciprofloxacin-mediated immunosuppression. J Immunol 184, 4827-4841.
  5. Kiessling M.K., Linke B., Brechmann M., Süss D., Krammer P.H. and Gülow K. (2010). Inhibition of NF-κB induces a switch from CD95L-dependent to CD95L-independent and JNK-mediated apoptosis in T cells. FEBS Lett 19;584(22):4679-88.
  6. Kiessling MK, Oberholzer PA, Mondal C, Karpova MB, Zipser MC, Lin WM, Girardi M, Macconaill LE, Kehoe SM, Hatton C, French LE, Garraway LA, Polier G, Süss D, Klemke CD, Krammer PH, Gülow K*, Dummer R*. (2011). High-throughput mutation profiling of CTCL samples reveals KRAS and NRAS mutations sensitizing tumors toward inhibition of the RAS/RAF/MEK signaling cascade. Blood 24;117(8):2433-40. (* shared last authorship)

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