Schematic of T cell development

αβ and γδ T lymphocytes comprise two distinct lineages that perform vital, non-overlapping roles in immune responses. These distinct lineages are thought to arise from a common thymic precursor, but despite much effort, very little is known about the developmental instructions that determine which of these lineages a developing thymocyte will adopt (Figure: Schematic of T cell development). Our laboratory seeks to gain insight into the molecular processes controlling alternate αβ/γδ lineage commitment. This is of critical importance not only because the divergence of T cells into functionally distinct αβ and γδ lineages is essential for normal immune responses, but also because differentiation of multipotential precursor cells into distinct cell types is a fundamental yet poorly understood process common to all multicellular organisms.

Role of TCR signal strength in fate adoption

Importantly, we recently provided compelling evidence for a signal strength model of lineage commitment which posits that weak signals promote commitment to the αβ lineage while comparatively strong signals promote commitment to the γδ lineage, irrespective of the TCR complex from which they originate. We further proposed that the differences in signal strength that alter fate are dependent upon differential activation of the signaling pathway consisting of: 1) the proximal signaling molecule, extracellular-signal regulated kinase (ERK); 2) downstream transcription factors of the early growth response (Egr) family; and 3) the Egr target, inhibitor of DNA-binding 3 (ID3) (Figure: Role of TCR signal strength in fate adoption).

Features of the KN6 γδTCR transgenic model

In pursuing these studies, we exploited an ideally suited γδTCR transgenic model (KN6), which has a known ligand whose expression can be manipulated to alter the nature of the resultant TCR signal. We have demonstrated that the KN6 γδ TCR complex requires engagement by ligands to promote adoption of the γδ fate (Figure: Features of the KN6 γδTCR transgenic model). This finding was highly controversial when first reported, but is now gaining support as other TCR/ligand pairs are examined. We are currently addressing the following important questions: 1) Do the TCR signals that control adoption of the αβ and γδ fate differ in intensity, duration, or both?, 2) What are the intracellular signaling pathways involved?, 3) How does altering the affinity of TCR-ligand interaction affect cell fate choice? and 4) Do different TCR signals actually direct lineage fate (instructive model), or do they weed out cells that have made the wrong choice (stochastic/selective model). In the latter model, strong TCR signals do not direct γδ development, but are essential for its completion, whilst preventing αβ development.

Blockade of αβ T cell development by Rpl22 deficiency

Mutations in ribosomal structural proteins or in proteins regulating ribosome assembly have been implicated in diseases such as Diamond-Blackfan Anemia and Dyskeratosis Congenita which are characterized by defects in hematopoiesis in the bone marrow; however, such mutations had never before been found to selectively affect development of T lineage cells. We have recently found that mice lacking the ubiquitously expressed ribosomal protein L22 (Rpl22) are grossly normal, but exhibit a strikingly specific defect in T cell development. Indeed, while Rpl22-deficiency had only a mild effect on development of γδ lineage T cells, it caused a profound and selective arrest of the development of αβ lineage T cells. The developmental arrest was accompanied by a significant increase in apoptosis among αβ lineage cells, which was caused by induction of p53 expression. Indeed, p53-deficiency blocked death and restored development of Rpl22-deficient thymocytes indicating that p53 is a critical target of Rpl22 regulation (Figure: Blockade of α/β T cell development by Rpl22-deficiency). Importantly, Rpl22-deficiency appears to induce p53 at least in part by increasing p53 synthesis. Taken together, these data indicate that Rpl22-deficiency activated a p53-dependent checkpoint that produced a remarkably selective block in αβ T cell development but spared γδ lineage cells, suggesting that some ribosomal proteins may perform cell-type or stage-specific functions. Efforts are currently underway to elucidate the molecular basis for selective induction of p53 in Rpl22-deficient αβ lineage progenitors by addressing the following questions: 1) How is Rpl22 expression differentially regulated in αβ and γδ lineage cells?;  2) Is p53 regulation by Rpl22 direct and if so to what sites in p53 mRNA does Rpl22 bind?; 3) How is p53 translation differentially regulated in αβ and γδ lineage progenitors?; and 4) How do the functions of the paralogous pair, Rpl22 and Rpl22-like 1, function to control hematopoiesis in zebrafish?

Ribosomal proteins are increasingly implicated in regulating cellular transformation, in some cases exhibiting tissue tropism. Nevertheless, it remains unclear how these widely expressed proteins can regulate development and transformation in a tissue restricted manner. Unlike other widely expressed ribosomal proteins whose germline ablation causes lethality, Rpl22-deficient mice exhibit a specific defect in development of αβ, but not γδ, T cells through cell-type specific translational-derepression of p53. We have recently extended this observation and revealed a unique role for Rpl22 in transformation. Indeed, loss of one allele of Rpl22 appears to predispose T lineage precursors to transformation (PICT5). Our hypothesis is that Rpl22 is an integral part of a signaling pathway that critically regulates T cell development and transformation. This may relate in part to the ability of Rpl22 to regulate cellular stress responses. We are currently attempting to determine the extent to which the Rpl22 gene is mutated in human cancer and whether such mutations serve as a biomarker of prognosis or therapeutic responsiveness. Indeed, we have found that a substantial fraction of T-ALL examined so far exhibit deletion of one Rpl22 allele and that these deletions are enriched in patients that succumb to the disease. While pediatric T-ALL is successfully treated in most cases, the prognosis for the remaining cases in which relapse occurs continues to be dismal. Identification of biomarkers predicting an aggressive disease course or predicting a pattern of drug responsiveness would be extremely useful in clinical management of the cases that do not respond to standard chemotherapy. Our current focus is to determine the mechanistic basis by which Rpl22 regulates transformation by addressing the following questions: 1) How does Rpl22 regulate stress responses and does this influence the development of cancer?; 2) What are the cellular targets whose expression is regulated by Rpl22 and how are they involved in transformation?

Because of the conservation of essential elements of hematopoiesis between zebrafish and man, we are undertaking a forward genetic screen in zebrafish to identify genes essential for normal T cell development. Details may be found at the web site of Dr. Jennifer Rhodes. The motivation for this effort is our hypothesis that genes that are essential for normal T cell development will also be involved in transformation. Studies on the Rpl22 gene described above are supportive of this idea.

Causes of SCID in humans have all been mapped to mutations that affect coding sequences. Efforts are ongoing to identify causes of SCID in patients by performing massively parallel sequencing of the exomes of SCID kindreds using the Illumina platform and then screening candidate genes for function in zebrafish. This represents yet another strategy with which to identify genes that are essential for normal immune cell development. Following identification, their role in the etiology or diagnosis of hematologic malignancies will be explored.