After repeated antigen exposure, both memory and terminally differentiated cells can be generated within CD8+ T cells. Although, during their differentiation, activated CD8+ T cells may first lose CD28, and CD28− cells may eventually express CD57 as a subsequent step, a population of CD28+CD57+(DP) CD8+ T cells can be identified in the peripheral blood. How this population is distinct from CD28−CD57−(DN) CD8+ T cells, and from the better characterized non‐activated/early‐activated CD28+CD57− and senescent‐like CD28−CD57+ CD8+ T cell subsets is currently unknown. Here, RNA expression of the four CD8+ T cell subsets isolated from human PBMCs was analyzed using microarrays. DN cells were more similar to “early” highly differentiated cells, with decreased TNF and IFN‐γ production, impaired DNA damage response and apoptosis. Conversely, increased apoptosis and expression of cytokines, co‐inhibitory, and chemokine receptors were found in DP cells. Higher levels of DP CD8+ T cells were observed 7 days after Hepatitis B vaccination, and decreased levels of DP cells were found in rheumatoid arthritis patients. More DP and DN CD8+ T cells were present in the bone marrow, in comparison with PBMCs. In summary, our results indicate that DP and DN cells are distinct CD8+ T cell subsets displaying defined properties.
We found recently that TNF-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor–inducible-14 (Fn14) by virtue of their strong capability to reduce the freely available cytoplasmic pool of TNFR-associated factor (TRAF)2 and cellular inhibitors of apoptosis (cIAPs) antagonize the functions of these molecules in TNFR1 signaling, resulting in sensitization for apoptosis and inhibition of classical NF-κB signaling. In this study, we demonstrate that priming of cells with TWEAK also interferes with activation of the classical NF-κB pathway by CD40. Likewise, there was strong inhibition of CD40 ligand (CD40L)–induced activation of MAPKs in TWEAK-primed cells. FACS analysis and CD40L binding studies revealed unchanged CD40 expression and normal CD40L–CD40 interaction in TWEAK-primed cells. CD40L immunoprecipitates, however, showed severely reduced amounts of CD40 and CD40-associated proteins, indicating impaired formation or reduced stability of CD40L–CD40 signaling complexes. The previously described inhibitory effect of TWEAK on TNFR1 signaling has been traced back to reduced activity of the TNFR1-associated TRAF2–cIAP1/2 ubiquitinase complex and did not affect the stability of the immunoprecipitable TNFR1 receptor complex. Thus, the inhibitory effect of TWEAK on CD40 signaling must be based at least partly on other mechanisms. In line with this, signaling by the CD40-related TRAF2-interacting receptor TNFR2 was also attenuated but still immunoprecipitable in TWEAK-primed cells. Collectively, we show that Fn14 activation by soluble TWEAK impairs CD40L–CD40 signaling complex formation and inhibits CD40 signaling and thus identify the Fn14-TWEAK system as a potential novel regulator of CD40-related cellular functions.
TWEAK, like many other ligands of the TNF family, occurs naturally in two forms, as a type II transmembrane protein and as soluble ligand released from the latter by proteases of the furin family. Both TWEAK variants interact with high affinity with Fn14, an unusual small member of the TNF receptor family. TWEAK and Fn14 activate a variety of intracellular signaling pathways but regulation of TNF-induced cell death and stimulation of the classical and alternative NFκB pathway are certainly the best understood ones. Intriguingly, soluble and membrane TWEAK significantly differ in their ability to trigger these responses. While activation of the alternative NFκB pathway and enhancement of TNF-induced cell death are efficiently induced by both forms of TWEAK, membrane TWEAK has a much higher capacity than soluble TWEAK to stimulate the classical NFκB pathway. Importantly, soluble TWEAK gains a membrane TWEAK-like Fn14 stimulating activity upon oligomerization or artificial anchoring to the cell surface. On the example of NFκB signaling and enhancement of TNF-induced cell death, we summarize here protocols that allow the identification of signaling pathways/cellular responses that preferentially respond to membrane TWEAK. These protocols base either on the side-by-side analysis of soluble TWEAK and oligomerized or cell surface-anchorable TWEAK variants or on the use of transfectants expressing soluble and membrane TWEAK.
Background: Obesity has been associated with chronic inflammation and oxidative stress. Both conditions play a determinant role in the pathogenesis of age-related diseases, such as immunosenescence. Adipose tissue can modulate the function of the immune system with the secretion of molecules influencing the phenotype of immune cells. The importance of the bone marrow (BM) in the maintenance of antigen-experienced adaptive immune cells has been documented in mice. Recently, some groups have investigated the survival of effector/ memory T cells in the human BM. Despite this, whether high body mass index (BMI) may affect immune cells in the BM and the production of molecules supporting the maintenance of these cells it is unknown. Methods: Using flow cytometry, the frequency and the phenotype of immune cell populations were measured in paired BM and PB samples obtained from persons with different BMI. Furthermore, the expression of BM cytokines was assessed. The influence of cytomegalovirus (CMV) on T cell subsets was additionally considered, dividing the donors into the CMV − and CMV + groups. Results: Our study suggests that increased BMI may affect both the maintenance and the phenotype of adaptive immune cells in the BM. While the BM levels of IL-15 and IL-6, supporting the survival of highly differentiated T cells, and oxygen radicals increased in overweight persons, the production of IFNγ and TNF by CD8 + T cells was reduced. In addition, the frequency of B cells and CD4 + T cells positively correlated with BMI in the BM of CMV − persons. Finally, the frequency of several T cell subsets, and the expression of senescence/exhaustion markers within these subpopulations, were affected by BMI. In particular, the levels of bona fide memory T cells may be reduced in overweight persons. Conclusion: Our work suggests that, in addition to aging and CMV, obesity may represent an additional risk factor for immunosenescence in adaptive immune cells. Metabolic interventions may help in improving the fitness of the immune system in the elderly.
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