Primary immunodeficiency (PID) is a term used to indicate disorders of the immune system that are genetically determined. PIDs are a group of rare and heterogeneous diseases that can affect all arms of the immune system and generally present with infection patterns unusual for severity, frequency, anatomical distribution and/or involved organisms. Other frequent clinical features of PIDs are susceptibility to autoimmune diseases, autoinflammatory syndromes and malignancy.
About 300 forms of PID are currently known the study of which represent unique opportunities to unravel the mechanisms underlying development, differentiation and function of the immune system.
Defining the molecular basis of PIDs
While many forms of PIDs have clinical presentations that are pathognomonic, in many cases different disease have overlapping and common clinical features. In addition, atypical presentations of well-known syndromes often complicate the clinical interpretation. Establishing a definitive genetic diagnosis is of paramount importance to make prognostic assessments and to provide adequate clinical management and genetic counseling to affected patients and families. The revolution of genomic analysis brought about by the development of high-throughput, next generation sequencing (NGS) methods has allowed tremendous progress in identifying mutations that cause undiagnosed childhood genetic diseases. It is now therefore possible to apply NGS platforms to the screening of genes responsible for known forms of PIDs, as well as of candidate genomic regions that may be implicated in the pathophysiology of still undefined immunologic defects.
We have established a platform for targeted gene sequencing of a library of >360 genes that are either known to be causing PID phenotypes or that have known associations with biological pathways implicated by genes that are known to be causing PID phenotypes. Genomic DNA of patients with clinical suspicion of PID will be analyzed using this approach and deleterious genomic sequences identified and characterized. To this aim our laboratory performs:
1. Studies in patient-derived primary immune cells.
Several immune system cell types (e.g. lymphocytes, dendritic cells) can be maintained in culture for relatively long period of time, which allows for characterization of functional defects in vitro. Examples of such functional assays are cell proliferation and cytokine production tests, cytotoxic and podosome formation assays, etc.1-12
2. Studies in patient-derived immortalized cell lines.
When possible and relevant, lymphocyte cell lines will be established through immortalization mediated by Epstein-Barr virus for B lymphocytes and saimiri virus or HTLV-1 for T lymphocytes.3,4,13-15
3. Studies in patient-derived primary progenitor cells.
Several in vitro differentiation systems are available that can be utilized to generate cell types that may be present in the patient’s circulation in small numbers or to analyze the differentiation steps that may be affected by the specific genetic defects. To these aims, hematopoietic progenitors cells obtained from patients are induced to differentiate into mature T, B, NK and myeloid cells in vitro.16-18
4. Studies in patient-derived induced pluripotent stem cells (iPSc).
Access to patient’s primary hematopoietic progenitors can be challenging. Generation of patient-derived iPSCs from patient dermal fibroblasts overcomes this potential problem. Fibroblasts will be reprogrammed into iPSCs through the temporary expression of OCT4, SOX2, KLF4 and cMYC, and differentiated into embryoid bodies that, in turn, will be used in the lympho-hematopoietic differentiation systems described above.
5. Modeling specific gene mutations by gene editing.
We will supplement the above-described studies with the generation of model systems based on healthy control cells in which the original variants detected in patient samples will be introduced by gene editing using zinc-finger nucleases and the CRISPR/Cas-9 system.19 Target cells types will include primary lymphocytes, hematopoietic progenitors (e.g. CD34+ bone marrow, cord blood or peripheral blood mobilized progenitors), and iPS cells.
Role of the Wiskott-Aldrich Syndrome Protein in the Differentiation and Regulation of the Immune System
The proposed research project plans to investigate the role of the Wiskott-Aldrich Syndrome protein (WASp) in the mechanisms that underlie the differentiation of the normal immune system and the maintenance of tolerance. Our experimental plan builds on prior observations from our group and demonstrating that both mice and humans with affected expression of WASp have important phenotypic and functional deficiencies within several key cellular components of the immune system (e.g. natural regulatory T cells - Treg, immature and mature B lymphocytes) that results in immunodeficiency, immune dysregulation and development signs and symptoms of autoimmunity.5,7-9,11-12,20-27 This project aims at expanding our studies to other critical aspects of immune system regulation such as the generation of peripherally induced regulatory T cells (iTreg), Treg cell-derived effector T lymphocytes (Ex-Treg cells), marginal zone (MZB) and follicular B cells, as well as follicular helper T lymphocytes (Tfh cells). Based on the current knowledge of the immunological role of WASp and our own published data, we have formulated the following main hypotheses:
1. WASp has a cell-intrinsic role in the function of FoxP3-expressing, regulatory T cells;
2. Wasp plays a role in the differentiation of MZB cells at the BCR and/or Notch2 signal level.
3. Wasp plays a role in the germinal center tolerance checkpoint in GC B cells and/or Tfh cells.
To test these hypotheses, we will pursue the following primary objectives:
a) To characterize a mouse model of regulatory T cell-specific WASp deficiency by performing T and B cell suppression assays, study the effects of the absence of WASp in the generation of iTregs, define the effects of Wasp-defective Tregs in the occurrence of peripheral autoreactive B cells, and investigate the effects of the absence of WASp on the the generation of effector Ex-Treg cells.
b) To assess the role of WASp in the BCR and NOTCH signal-dependent MZB cell differentiation and maintenance steps in inducible B cell conditional Was knock-out mice.
c) Assess the role of WASp in peripheral naïve B cell tolerance and GC B cell selection.
d) Assess the susceptibility to induced and spontaneous autoimmunity of mice carrying WASp deficient Tfh cells.
The results deriving from this proposal will advance our understanding of the mechanisms underlying the differentiation of the normal immune system, the maintenance of tolerance and have the potential to lead to the development of novel therapeutics for autoimmune diseases.
This research project investigates the role of the Wiskott-Aldrich Syndrome protein (WASp) in the immunological mechanisms that are responsible for maintenance of tolerance. Genetic deficiency of WASp expression in humans results in a clinical syndrome including thrombocytopenia, hemorrhages, eczema, recurrent infections, cancer, and autoimmunity. The latter complication can affect 40% to 70% of the patients and its occurrence has been associated with increased susceptibility to malignancies and poor overall prognosis.
Prior observations from our group demonstrated that both mice and humans with defective or absent expression of WASp have important functional deficiencies among critical tolerance inducing immune cells, such natural regulatory T cells (nTreg) 5,9 and develop signs and symptoms of autoimmunity 11,20. Our current research aims at expanding our investigations to other critical aspects of immune system regulation such as the generation of peripherally induced regulatory T cells (iTreg), generation and function of regulatory B cells (Breg) and transcriptional regulation of Th1 differentiation. Based on the current knowledge of the immunological role of WASp, our own published and preliminary data, we have formulated the following main hypotheses:
To test these hypotheses, we are pursuing the following objectives:
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3 Wada, T., Jagadeesh, G. J., Nelson, D. L. & Candotti, F. Retrovirus-mediated WASP gene transfer corrects Wiskott-Aldrich syndrome T-cell dysfunction. Hum Gene Ther13, 1039-1046 (2002).
4 Bosticardo, M. et al. Retroviral-mediated gene transfer restores IL-12 and IL-23 signaling pathways in T cells from IL-12 receptor beta1-deficient patients. Mol Ther9, 895-901 (2004).
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8 Taylor, M. D. et al. Nuclear role of WASp in the pathogenesis of dysregulated TH1 immunity in human Wiskott-Aldrich syndrome. Sci Transl Med2, 37ra44 (2010).
9 Adriani, M. et al. Defective inhibition of B-cell proliferation by Wiskott-Aldrich syndrome protein-deficient regulatory T cells. Blood117, 6608-6611 (2011).
10 Lawrence, M. G. et al. Elevated IgE and atopy in patients treated for early-onset ADA-SCID. J Allergy Clin Immunol (2013).
11 Shimizu, M. et al. Aberrant glycosylation of IgA in Wiskott-Aldrich syndrome and X-linked thrombocytopenia. J Allergy Clin Immunol131, 587-590 e581-583 (2013).
12 Simon, K. L. et al. Molecular and phenotypic abnormalities of B lymphocytes in patients with Wiskott-Aldrich syndrome. J Allergy Clin Immunol133, 896-899 e894 (2014).
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16 Haddad, R. et al. Dynamics of thymus-colonizing cells during human development. Immunity24, 217-230 (2006).
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18 Lagresle-Peyrou, C. et al. Human adenylate kinase 2 deficiency causes a profound hematopoietic defect associated with sensorineural deafness. Nat Genet41, 106-111 (2009).
19 Peterson, L. et al. Comparison of Gene Editing Strategies for Gene Correction of Wiskott-Aldrich Syndrome in Mouse Embryonic Stem Cells. Mol. Ther.22, S216-S217 (2014).
20 Shimizu, M. et al. Development of IgA nephropathy-like glomerulonephritis associated with Wiskott-Aldrich syndrome protein deficiency. Clin Immunol142, 160-166 (2012).
21 Recher, M., et al., B cell-intrinsic deficiency of the Wiskott-Aldrich syndrome protein (WASp) causes severe abnormalities of the peripheral B-cell compartment in mice. Blood119, 2819-2828 (2012).
22 Crestani, E., et al., Broad spectrum of autoantibodies in patients with Wiskott-Aldrich syndrome and X-linked thrombocytopenia. J Allergy Clin Immunol136, 1401-1404 (2015).
23 Kolhatkar, N.S., et al., Altered BCR and TLR signals promote enhanced positive selection of autoreactive transitional B cells in Wiskott-Aldrich syndrome. J Exp Med212, 1663-1677 (2015).
24 Pala, F., et al., Lentiviral-mediated gene therapy restores B cell tolerance in Wiskott-Aldrich syndrome patients. J Clin Invest125, 3941-3951 (2015).
25 Yokoyama, T., et al., Age-Dependent Defects of Regulatory B Cells in Wiskott-Aldrich Syndrome Gene Knockout Mice. PLoS One 10, e0139729 (2015).
26 Volpi, S., et al., N-WASP is required for B-cell-mediated autoimmunity in Wiskott-Aldrich syndrome. Blood 127, 216-220 (2016).
27 Lexmond, W.S., et al., FOXP3+ Tregs require WASP to restrain Th2-mediated food allergy. J Clin Invest 126, 4030-4044 (2016).