This project consists of the creation of important tools for measuring T cell receptor diversity and TCR-mediated signaling, and use of these tools in both patients and experimental models of TCR diversity. The first tool is the use of high-throughput sequencing to measure the diversity of T-cell receptor rearrangements. The strategies and protocol development are ongoing and being done in collaboration with Danny Douek's lab in the NIH VRC. Finding 1: The next generation-based TCR sequencing method and techniques for analysis are completed. We applied this method to the study of patients with rare diseases of "leaky" severe combined immune deficiency. These patients have mutations in genes known to cause SCID, but either have a profound lymphoproliferative disorder associated with severe dermatitis, elevated IgE and eosinophilia, called Omenn syndrome, or a milder phenotype, associated with milder effects on protein activity, characterized by autoimmunity and granulomatous disease, called hypomorphic SCID. We found that patients with Omenns syndrome due to mutations in recombinase activating gene (Rag) had marked repertoire constriction, substantially smaller CDR3 regions and impaired N-nucleotide addition compared to those with Omenn syndrome due to other causes, suggesting that normal Rag function is critical for T-cell receptor junctional diversity. We also found that while the TCR repertoire diversity in the hypomorphic SCID patients was similar to controls, and while amino acid usage was similar to controls in Omenn Syndrome patients, there were significantly different residues used in the hypomorphic SCID, arguing for a specific set of TCRs which are pathogenic in their disease and not in a disease characterized by even more profound repertoire reduction. The results of this study are currently under review for publication. Finding 2: We have developed a functional screen for TCR diversity using peptides generated by random amino acids at every position except one. These 15-mer peptides provide a very diverse peptide pool, within which we believe are sufficient numbers to lead to activation of a proportion of T cells in normal, polyclonal T cells samples. By creating individual libraries with only one fixed amino acid at a total of 3 different positions, we created a test panel of 66 peptide libraries. Each peptide library was then added to a different well of PBMC cultures of a normal volunteer, and then assayed for upregulation of CD69 and CD98 after 24 hours, or for dilution of CFSE after one week. When the panel was used on multiple normal donors, there was excellent correlation between those peptide libraries which lead to upregulation of CD69/CD98 and to CFSE dilution. The activation was dose-dependent and was blocked by MHC Class II blockade, arguing that the peptides were acting via TCR-MHC binding. We have used this tool to show that the naive repertoires of T cell receptors are constricted in aged patients compared to younger patients, that naive T-cells have broader reactivity than memory T-cells and that central memory T-cells in the hyper-IgE syndrome have a marked reduction in TCR reactivity. In addition, we can measure the correlation between regulatory and effector CD4 T cell repertoires using this tool. We have found that, while healthy individuals have a very tight correlation between the two, patients that have certain lymphopenic conditions (ADA-SCID and Idiopathic CD4 lymphopenia) with strong allergic phenotypes actually lose this correlation. We believe the lack of correlation between the TCR specificities of effector and regulatory T cells explains the allergic phenotypes these patients develop, and our method is one of the only ways this can be detected. We then found that this technique can also be used to prime naive T cells by mixing sorted, naive CD4+ cells with peptide libraries loaded onto autologous DC generated from the donors monocytes. Again, the stimulation was measured by CFSE dilution, could be inhibited by MHC Class II blockade, and most interestingly, could be modulated to create different Thelper phenotypes. Specifically, naive CD4+ T cells could be differentiated into Th1 and Th2 cells. Low peptide doses at priming, independent of exogenous Th1 or Th2 conditions, predisposed towards Th2 differentiation, while high peptide doses led to Th1 differentiation. This system allows for the study of polyclonal human naive T cell responses to peptide stimulation and priming. The manuscript describing these findings was published in the Proceedings of the National Academy of Sciences, USA. We have also used this screening system to identify a patient with a different T cell receptor signaling mutation. The patient was an infant with severe eczema, elevated IgE and multiple food allergies. He also had a few syndromic features such as mild pulmonic stenosis and undescended testes. ERK phosphorylation in the patient showed a rapid early peak with rapid decrease compared to control, while early GATA3 expression within his naive T cell compartment was elevated and not blocked by IL-4 blockade. Because of the pulmonic stenosis, SOS1 was sequenced, as mutations in this gene are seen in patients with Noonan syndrome, which can be characterized by some of the features seen in this patient. SOS1 is a member of the Ras/MapK pathway and mutations in this pathway can cause Noonan, LEOPARD, Cardiofaciocutaneous, and Costello Syndromes - a group of multisystem congenital disorders with significant variability, but most often associated with cardiac, neurodevelopmental and skeletal manifestations. Of interest, there are reports in the literature of allergy and immune deficiency in a subset of these patients, although they have not been well characterized, nor has the impact of this class of mutations on T cell function ever been measured. The mutations appear to lead to gain-of-function of the Ras/Map Kinase pathway, and mouse models have shown that many symptoms of these disorders can be cured with MEK inhibition. Lymphocyte signaling and function have not been studied in these patients, raising the question as to whether mutations in this pathway might lead to abnormal T cell differentiation and effector function. Finding 3: Patients with classic ADA-SCID have lymphopenia and restricted repertoires. We therefore wished to examine Th2 cytokine production and allergic disease in a cohort of 19 patients being treated for ADA-SCID. We found a profound increase in Th2 cytokine production by T cells in all these patients, with no effect on Th1 cells. Treg percentages were normal, but T-cell activation was impaired. At the same time, allergic disease including marked serum IgE elevation was present in 50% of the patients, and did not correlate to Th2 cytokine production. Because non-allergic patients were younger, it is quite possible that the allergic predisposition is present in all patients with classic ADA-SCID, as evidenced by the Th2 cytokine production, and that clinical allergy may emerge in the majority of them as they age. These results have been published in the Journal of Allergy and Clinical Immunology.
|Yu, Xiaomin; Almeida, Jorge R; Darko, Sam et al. (2014) Human syndromes of immunodeficiency and dysregulation are characterized by distinct defects in T-cell receptor repertoire development. J Allergy Clin Immunol 133:1109-15|
|Lawrence, Monica G; Barber, John S; Sokolic, Robert A et al. (2013) Elevated IgE and atopy in patients treated for early-onset ADA-SCID. J Allergy Clin Immunol :|
|Barber, John S; Yokomizo, Lauren K; Sheikh, Virginia et al. (2013) Peptide library-based evaluation of T-cell receptor breadth detects defects in global and regulatory activation in human immunologic diseases. Proc Natl Acad Sci U S A 110:8164-9|
|Datta, Shrimati; Milner, Joshua D (2011) Altered T-cell receptor signaling in the pathogenesis of allergic disease. J Allergy Clin Immunol 127:351-4|