Tuesday, December 20, 2011

Human RAG deficiency: not just immunodeficiency

Novel insights into mechanisms of immune dysregulation: research of Jolan E. Walter, MD, PhD

Jolan E. Walter, MD, PhD

Jolan E. Walter, MD, PhD
Clinical Instructor in Pediatrics, Harvard Medical School; Physician, Pediatric Allergy/Immunology, MassGeneral Hospital for Children

Mentors: Luigi Notarangelo MD, Shiv Pillai, MD, PhD.

It is becoming increasingly recognized that mutations in SCID-associated genes may result in significant phenotypic variability. In particular, mutations of the RAG genes in humans have been associated with T- B- NK+ SCID, Omenn syndrome, leaky SCID, CID with expansion of gd T cells, delayed-onset immunodeficiency with granuloma, and idiopathic CD4 lymphopenia.(Figure 1)  Recently a novel flow-cytometric assay was developed in which Rag1-/- tg.bcl2 abelson-transformed pro-B cell line containing  a single integrant with an inverted GFP cassette flanked by Recombination Signal Sequences (RSS) are infected with retroviral vector expressing wild-type or mutant human RAG1. In this system, GFP expression is used as a read-out of RAG activity. With the help of the assay a series of more than 40 different RAG1 mutants analyzed, a tight correlation has been observed between degree of residual activity and clinical/ immunological phenotype. Of note, mutations associated with Omenn syndrome have very low activity (0.5-2% of wild-type), whereas mutations associated with the mildest forms of disease (idiopathic CD4 lymphopenia and granuloma formation) carry significant residual activity (up to 40-60% of wild-type) on one allele.

These data support genotype-phenotype correlation in RAG deficiency, so that hypomorphic mutations may allow for residual T and B cell development and hence result in a phenotype other than T- B- SCID. However, It is now recognized that an important feature of immunodeficiency associated with hypomorphic RAG mutations is represented by the frequent occurrence of autoimmunity. In particular, RAG mutations  with very low V(D)J recombination activity are associated with leaky SCID and CID with expansion of γδ T cells, in which autoimmune cytopenias are common. On the other hand, patients with RAG mutations with higher functional activity often present with CID + granuloma, and tend to develop severe autoimmune manifestations including myasthenia gravis, seronegative vasculitis, vitiligo and Guillain-Barre syndrome.

The exact pathomechanisms of B cell dysregulation in RAG deficiencies is still unclear. Impaired (but not abolished) RAG activity is expected to significantly reduce receptor editing and to associate with high serum levels of BAFF because of B cell lymphopenia. Both abnormalities have been confirmed in a mouse model of leaky SCID due to a homozygous hypomorphic Rag1 mutation (S723C), and may cause increased export and survival of self-reactive B lymphocytes.(Figure 2) However, analysis of B-cell mediated autoimmunity in patients with hypomorphic RAG mutations has received little attention. Using an autoantibody capture array, we have shown that elevated levels of autoantibodies may be seen in a proportion of patients with hypomorphic RAG mutations, and in Rag1 S723C homozygous mice.  These autoantibodies also include a discrete set of anti-cytokine antibodies.

Finally, significant variability of the clinical and immunological phenotype has been reported even among patients carrying the same RAG defect. It has been hypothesized that environmental factors may act as disease modifiers, as best exemplified by RAG-deficient patients whose initial T- B- SCID phenotype has turned into Omenn syndrome of CID + gd T cells at the time of viral infections. We have investigated the role of innate immunity-mediated signals in this phenomenon. Rag1 S723C mice die rapidly when injected i.v. with poly(I:C).(Figure 3) Furthermore, repeated i.p. injections of lower doses of poly(I:C result in dramatic increase of the titer and diversity of autoantibodies produced in vivo

These data provide mechanistic insights into the immune dysregulation of the Rag-dependent primary immunodeficiencies.




1. Pessach I, Walter JE, Notarangelo L. Recent advances in Primary Immunodeficiencies: identification of novel genetic defects and unanticipated phenotypes. Pediatric Research 2009 Jan 28. (PMID: 19190530)

2. Rucci F, Notarangelo LD, Fazeli A, Partizi L, Hickernell T, Paganini T, Coakley K, Detre C, Keszei M, Walter JE, Feldman L, Cheng HL, Poliani L, Wang JH, Balter B, Recher M, Andersson E, Zha S, Giliani S, Terhorst C, Alt FW, Yan C. Homozygous DNA Ligase IV R278H mutation in mice leads to leaky SCID and represents model for human LIG4 syndrome. Proc Natl Acad Sci USA. 2010 Feb (PMID: 20133615)

3. Walter JE, Rucci F, Detre C, Patrizi L, Paganini T, Recher M, Keszei M, Pessach I, Lang PA, Regenass S, Giliani S, Andersson E, Sekiguchi J, Al-Herz W, Cowan MJ, Dbaibo G, ElGhazali G, Gennery AR, Pasic S, Puck JM, Malech H, DeRavin SS, Niehues T, Schuetz C, Bleesing J, Terhorst C, Alt FW, Notarangelo LD. Defects of B cell tolerance and expansion of immunoglobulin-secreting cells in rag-dependent immunodeficiency Journal of Experimental Medicine 2010 July (PMID: 20547827)

4. de la Fuente MA, Recher M, Rider NL, Strauss KA, Morton DH, Adair M, Bonilla FA, Ochs HD, Gelfand EW, Pessach IM, Walter JE, King A, Giliani S, Pai SY, Notarangelo LD. Reduced thymic output, cell cycle abnormalities, and increased apoptosis of T lymphocytes in patients with cartilage-hair hypoplasia. J Allergy Clin Immunol 2011 July (PMID: 21570718)

5. Meyers G, Ng YS, Bannock JM, Lavoie A, Walter JE, Notarangelo LD, Kilic SS, Aksu G, Debré M, Rieux-Laucat F, Conley ME, Cunningham-Rundles C, Durandy A, Meffre E. Activation-induced cytidine deaminase (AID) is required for B-cell tolerance in humans.Proc Natl Acad Sci USA 2011 July  (PMID: 21700883)

6. Recher M, Berglund LJ, Avery DT, Cowan MJ, Gennery AR, Smart J, Peake J, Wong M, Pai SY, Baxi S, Walter JE, Palendira U, Tangye GA, Rice M, Brothers S, Al-Herz W, Oettgen H, Eibel H, Puck JM, Cattaneo F, Ziegler JB, Giliani S, Tangye SG, Notarangelo LD. IL-21 is the primary common gamma chain-binding cytokine required for human B-cell differentiation in vivo. Blood 2011 (PMID: 22039266)

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