Estrategias audición para desarrollar la comprensión lectora

I spent about three years studying thymopoiesis and another three years

investigating the biochemical and functional interaction between Foxp3 and Siva. At the end, I am most curious about Siva functions that may be Foxp3-independent. Does Siva affect thymopoiesis or hematopoiesis? Could Siva somehow be implicated in hyper immune activation associated with CD70:CD27 signaling? Answers to some of these questions could inform future investigations regarding a putative functional interaction between Foxp3 and Siva.

SIVA AND THYMOPOIESIS

The question of Siva’s potential role in thymopoiesis was first raised by the observation that double positive (DP) thymocytes express high levels of Siva protein9. Since then, no reports about Siva function in the thymus have been published.

Siva is pro-apoptotic and thymopoiesis is a highly selective process that relies on apoptosis to remove around 97% of thymocytes initiated. Other pro-apoptotic factors are known to sensitize thymocytes to death by neglect.

Siva’s pro-apoptotic function is associated with Bcl-2 family members and these factors are associated with various thymopoiesis checkpoints. Siva promotes

mitochondrial-dependent apoptosis by binding and inhibiting Bcl-2 and Bcl-XL. Bcl-2 overexpression rescues thymopoiesis in IL-7 signaling-deficient mice, but neither Bcl-2 nor Bcl-XL is required for thymopoiesis. Instead, the pro-survival Bcl-2 family member, Mcl-1 provides an essential survival signal during thymopoiesis. Mcl-1 deficient mice arrest at the DN stage and Siva is highly expressed at the subsequent DP stage, provoking the speculation that the balance between Siva and Mcl-1 contributes to TCRβ selection.

The region of Bcl-2/Bcl-XL required to interact with Siva has not been identified, so domain information cannot be used to predict whether Mcl-1 and Siva might

physically interact. Anti-apoptotic Bcl-2 and Bcl-XL each contain BH (Bcl-2 homology) domains 1-4 and a transmembrane domain (TMD). Pro-apoptotic Bax contains a TMD and BH domains 1-3. Bax does not interact with Siva, which invites the hypothesis that the BH4 domain could mediate Siva-binding. Mcl-1 is the only known pro-survival Bcl-2 family member that lacks a transmembrane domain; it contains BH1 and BH3 domains9- 13_.

Based on the known pro-apoptotic function of Siva, I would predict that thymocyte-specific knockdown should positively affect thymocyte number and negatively affect DP thymocyte deletion. This question could be tested in the FTOC

system described in Chapter Three. Given the complex relationship between different thymic lymphoid and stromal cell populations, I would investigate Siva’s effect on thymocyte distribution by analyzing all known thymic lymphoid populations (double negative, double positive, CD4 single positive, CD8 single positive, Treg, natural killer T cells, and γδ T cells). Also, I would perform histology to evaluate thymic cortico-

medullary organization.

In addition to possibly contributing to thymopoiesis, gene array data suggests that Siva could be involved in hematopoiesis separate from thymopoiesis. The mouse BioGPS gene atlas showed Siva expression to be extremely high in stem cell lines and in

hematopoietic progenitor cells compared to other cell populations assayed14,15. Myelopoiesis can be induced in vitro by adding cytokines and growth factors to the culture media. NK, B, and T cell development requires cytokines and stromal cell support. OP9 stromal cells support NK and B cell development because M-CSF is absent16. Notch ligand, expression (DL1 or DL4) enables OP9 cells to support T cell development, though not past the DP stage17. Therefore, well-characterized experimental systems are available that could be used to study Siva’s contribution to the differentiation of multiple HSC lineages.

CD27,SIVA EXPRESSION AND TCELL APOPTOSIS

Siva was first identified as a binding partner for the CD27 co-receptor, but a functional interaction between CD27 and Siva has not yet been observed in T cells under physiological conditions. Siva overexpression in HPB-ALL T lymphocytes enhanced apoptosis in response to CD27 ligation by CD70 in vitro18. Transgenic mouse data and

adoptive transfer experiments associate the CD27:CD70 axis with chronic T cell

activation19. In vitro, Siva negatively regulates T cells by inhibiting NFκB and IL-2, and by enhancing apoptosis20,21. Thus, in vitro data predicts that, in the presence of Siva, ligation of CD27 by CD70 would promote apoptosis and inhibit T cell activation. Instead, in vivo blockade of CD70 protects mice from colitis and arthritis.

One explanation that could resolve Siva in vitro data with CD27:CD70 in vivo data is inhibition of Siva by ubiquitin-mediated proteasomal degradation. LPA2 (Edg4), a G-protein coupled growth factor receptor and XIAP, an anti-apoptotic E3 ligase, both inhibited Siva function by ubiquitination and proteasome-dependent mechanisms in vitro6,20. LPA2-mediated Siva degradation required LPA stimulation. Growth factor withdrawal restored Siva expression6. Though the LPA2 experiments were not performed in a lymphocyte line, LPA2 is expressed by T cells and has been shown to mediate T cell migration7_{. A possible physiological scenario could exist where LPA supports survival of} CD27pos T cells by inhibiting Siva expression. Support for such a possibility requires more data pertaining to the expression kinetics and immunobiology of LPA, LPA2, Siva, CD27, and CD70.