Role of Notch Signaling in T-cell Development in Thymus

Background: DiGeorge Syndrome

DiGeorge syndrome is a congenital disease affecting infant’s immune system. The syndrome is classified by the absence or underdevelopment of the thymus and parathyroid glands, due to a large deletion on chromosome 22. Depending on the extent of the missing thymus and parathyroid tissue, the symptoms vary. Children who survive with DiGeorge syndrome live having defective immune systems. They are not able to develop proper adaptive immune systems, and are subjective to repeated infections (MayoClinic.com).

The thymus is important in supporting the differentiation and selection of T cells. It plays a crucial role in inducing the differentiation of bone marrow derived precursor cells to different types of T cells. In the thymus, the progenitor cells receive a signal through Notch1 receptors, which instruct the precursor cells to commit to the T-cell lineage rather than the B-cell lineage. Recent experiments have shown Notch signaling to be required throughout T cell development: T cell lineage commitment, alpha/beta versus gamma/delta t cell lineage commitment,  and CD4+ versus CD8+ lineage commitment (Murphy et al. 2009). Here we see how Notch signaling plays a role in T cell commitment.

Introduction: T cell Development

The T cell lineage is derived from hematopoietic stem cells that arise from bone marrow. During the T cell development the pluripotent hematopoietic stem cells in the marrow migrate to the thymus through the blood. Below is the diagram of the T cell lineage from hematopoietic stem cell:

Figure 1: Differentiation of pluripotent hematopoietic stem cell to various immune cells.  (Murphy et al. 2009)

Figure 2: Overview of the differentiation of early T-lineage precursor (ETP) to T cell.  (Labrique et al. 2011).

T-cell Development

Recent experiments have shown that Notch signaling pathways to be involved in determining T cell fates.  Generally, mammalian Notch proteins interact with ligands belonging to the Jagged family and Delta-like family. The interaction results in proteolytic cleavage of Notch either within or close to its transmembrane domain, which results in the release of intracellular domain of Notch. Notch moves to the nucleus interacting with CBF1 transcription factor, and converting it from a repressor to an activator of gene transcription.

Notch signaling: T cell lineage commitment

Experiments done by Radtke et al. 1999, show that induced deletion of Notch-1 in new born mice resulted in reduced size of thymus and number of subsets of T cells.

Figure 3: Abnormal thymus size and morphology in iNotch1 -/- mice. (A) The thymus isolated from 4-week-old Notch1lox/lox (control) and Notch1lox/lox MxCre1/2 mice, injected with IFN alpha. (B) Comparison of thymocyte number in two groups of mice, where Notch1 -/- show a 5 fold reduction in thymocyte number (Radtke et al. 1999).

When the cytofluorometric analysis of the CD4, CD8, and T cell receptor was performed on thymocytes of normal and Notch deficient mice, the Notch deficient mice showed a moderate decrease in mature single positive T cells (CD4 SP and CD8 SP), a relatively large decrease in CD4+8+ double positive (DP) cells and a small increase in CD4-8- double-negative (CN) T cells (Figure 4). Also TCR gamma/delta double negative cells were decreased in 10 fold and immature single positive thymocytes decreased 13-fold compared to controls.

Figure 4: Reduction of T cell subsets in the Thymus of iNotch1 -/- Mice. (A) FACS analysis of CD4 versus CD8 cells and histogram for TCT alpha/beta expression for total thymocytes. (B) Bar graph representing the absolute cell numbers for thymocyte subsets (Radtke et al. 1999).

Further experiments showed the levels of triple negative cells (CD25- CD44+ Cd4- Cd8-) in the thymus to be normal. However, these cells were shown to be expressing B cell markers and phenotypically similar to immature bone marrow B cells. These results showed that Notch signaling is required for differentiation of lymphoid progenitor cells to T cell lineage, and in the absence of Notch signaling lymphoid progenitor cells in the thymus were shown to differentiate to B cell lineage (Figure 5D).

Figure 5: Competitive repopulation of lethally irradiated C57/Bl6 mice reveals a complete inability of iNotch1 -/- bone marrow to generate T cells. (A) FACS profile of lymph node cells stained with anti-CD45.1, anti-CD45.2, anti-B220, and anti-Cd3 antibodies. (B) FACS profile of thymocytes stained with anti-CD45.1, anti-Cd45.2, anti-Cd4, andti-CD8, and anti-B220 antibodies (Radtke et al. 1999).

Further experiments done by Pui et al. 1999, showed Notch signaling to be sufficient to induce T cell lineage commitment. Bone marrow stem cells that expressed Notch-1 transferred to irradiated hosts, developed thymus independent cells with T cell markers, including Cd4, Cd8 and Thy-1 (Figure 6). They also showed that expression of Notch1 completely inhibits the cells differentiation into the B cell lineage, suggesting that Notch signaling blocks the differentiation of lymphoid progenitor cells into the B cell lineage and enables the cells to differentiate into the T cell lineage (Figure not shown here).

Figure 6: ICN1 expression causes the appearance of an immature T cell population in bone marrow. (A) GFP expression in BM and thymus 22–23 days after receiving syngeneic BM transduced with MigR1, Mig ICN1, or Mig DANK. (B) Flow cytometric analysis of BM from BALB/c mice 22–23 days after receiving syngeneic BM transduced with MigR1, Mig ICN1, or Mig DANK. (C) Oligoclonal TCRb rearrangements are detected at day 23 postBMT in the BM of mice receiving Mig ICN1–transduced BM cells. Arrowheads indicate the rearranged fragments (Pui et al. 1999).

Conclusion

Pui et. al have shown Notch signaling to play an important role in T cell lineage commitment by comparing the results from wild-type mice to the mutant mice that had knockouts of Notch1. However, by doing so it may have caused dis-regulated signaling, leading to confound interpretations (Yuan et al. 2010). Further experiments can be done to support such conclusions by doing genetic disruption of Notch receptors, ligands and/or signaling molecules.

References

“DiGeorge Syndrome – MayoClinic.com.” Mayo Clinic. 8 Aug. 2009. Web. 29 Apr. 2011. <http://www.mayoclinic.com/health/digeorge-syndrome/DS00998>.

Kebbeth, Murphy, Paul, Travers, and Walport, Mark. Janeway’s Immunobiology, 7ed. (Garland Science 2008)

Labrecque, Nathalie, Troy Baldwin, and Sylvie Lesage. “Molecular and Genetic Parameters Defining T-cell Clonal Selection.” Immunology & Cell Biology, 89.1 (2011): 16-26.

Michael L Deftos, Michael J Bevan, Notch signaling in T cell development, Current Opinion in Immunology, Volume 12, Issue 2, 1 April 2000, Pages 166-172, ISSN 0952-7915, DOI: 10.1016/S0952-7915(99)00067-9.(http://www.sciencedirect.com/science/article/B6VS1-3YT4419-7/2/bf9f6fe431f0bcc216cbf17dcf47c877)

Pui, JC, D Allman, LW Xu, S DeRocco, FG Karnell, S Bakkour, JY Lee, T Kadesch, RR Hardy, JC Aster, and WS Pear. “Notch1 Expression in Early Lymphopoiesis Influences B Versus T Lineage Determination.” IMMUNITY, 11.3 (1999): 299-308.

Radtke, F, A Wilson, G Stark, M Bauer, J van Meerwijk, HR MacDonald, and M Aguet. “Deficient T Cell Fate Specification in Mice with an Induced Inactivation of Notch1.” IMMUNITY, 10.5 (1999): 547-558.

Yuan JS, Kousis PC, Suliman S, Visan I, Guidos CJ. Functions of notch signaling in the immune system: consensus and controversies. Annu Rev Immunol. 2010 Mar;28:343-65. Review. PubMed PMID: 20192807.


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