Recombination-activating genes ( RAG) 1 and RAG2 encode endonuclease proteins, which are critical to initiate the molecular processes that lead to lymphocyte receptor formation. Nonsense mutations in RAG1/ RAG2 completely annul this process and abrogate T- and B-lymphocyte receptor formation, leading to the most profound immunodeficiency syndrome, severe combined immunodeficiency (SCID). A spectrum of less-severe clinical phenotypes is now recognized to be due to mutations in RAG genes; recent understanding of the impact of mutations on protein function helps to explain, at least some of, these features. This article summarizes recent findings and places the genetic and molecular findings in a clinical context.

The recombination activity retained by RAG mutants correlates with the severity of clinical presentation, in that the least recombination activity is associated with the most-severe phenotype.

T- and B-lymphocytes, key cells of the adaptive immune response, are generated in the thymus and bone marrow, respectively. Each naive lymphocyte has a unique antigen receptor and lymphocyte receptor diversity, generated by stochastic genetic rearrangements that cut, splice, and modify lymphocyte receptor variable region gene segments at the lymphocyte receptor loci, that is estimated to be in the region of 10 ²⁰ α–β chain combinations for T-lymphocyte receptors ³⁴ or heavy-light chain combinations for B-lymphocyte receptors.

DNA double-strand breaks introduced by an endonuclease complex coded by RAG1 and RAG2, which randomly incise DNA at highly conserved sequences of DNA (recombination signal sequences) that flank all variable (V), diversity (D), and joining (J) coding regions, initiate T-lymphocyte receptor gene segment re-arrangement in the thymic cortex, enabling the assembly of interspersed V(D)J gene elements, a process known as V(D)J recombination.

The stochastic re-assembly of V(D)J gene segments leads to the formation of a wide receptor repertoire. The random nature of receptor formation inevitably generates some strongly self-reactive lymphocyte receptors, which carry the risk of initiating autoimmune responses. Developing thymocytes that successfully re-arrange the antigen receptor therefore navigate a rigorous intra-thymic two-stage selection process to identify and remove these potentially damaging self-reactive T-lymphocytes ³⁵ . In the first phase, known as positive selection, thymocytes are exposed to a self-peptide/major histocompatibility complex (MHC) presented by cortical thymic epithelial cells in the thymic cortex. By recognition of this complex with intermediate receptor affinity, thymocytes can advance to the next developmental phase, ensuring that antigen is recognized in the company of self-MHC molecules. Receptors that fail to bind the complex or bind with high affinity facilitate apoptosis of the thymocyte.

Surviving thymocytes migrate to the thymic medulla and submit to the second stage of thymocyte selection, designated negative selection, during which the antigen receptor interacts with a self-peptide/MHC complex presented by medullary thymic epithelial cells and dendritic cells. Medullary thymic epithelial cells express a wide range of ectopic peripheral tissue-restricted self-antigens (TRA), partly controlled by the Autoimmune Regulator (AIRE) and Fezf2 transcription factors ^{36– 38} , exposing a “molecular mirror of peripheral self”. Antigen receptors that bind with high affinity to the TRA/MHC complexes are deleted, or forced down a regulatory T-lymphocyte developmental pathway, as otherwise these have the potential to elicit autoimmunity ³⁹ . Negative selection is an indispensable function of central tolerance, a key process that eliminates auto-reactive T-lymphocytes.

A calm and uninterrupted thymic microenvironment is required for normal T-lymphocyte development ⁴⁰ ; however, typical thymic architectural development relies on “thymic crosstalk”, i.e. feedback from the developing thymocytes ⁴¹ . CD4+ thymocytes express CD40 ligand and RANK ligand, which provide signals to medullary thymic epithelial cells ⁴² , enabling cellular maturation and subsequent expression of AIRE.

Seminal studies in infants with genetic defects that severely impair T-lymphocyte development have shown disrupted thymic architecture, with diminished thymic epithelial cell development, absent AIRE expression, and severely reduced FOXP3+ regulatory T-lymphocytes not seen in mutations that permit T-lymphocyte development ⁴³ . Furthermore, disturbed AIRE expression with diminished self-antigen expression has been demonstrated in the thymus of infants with hypomorphic RAG mutations ⁴⁴ . These findings demonstrate that significant impairment of T-lymphocyte development may diminish central and peripheral tolerance mechanisms, increasing the risk of escape of highly autoreactive T-lymphocyte clones and thus the risk of developing autoimmunity for an affected individual. Nonsense mutations in RAG1 or RAG2 disrupt the function of RAG endonucleases profoundly and result in complete failure to initiate antigen receptor V(D)J re-arrangement, which causes failure of progression of T- and B-lymphocyte progenitors beyond the DN3 and pre-B-1 stage of development. This gives rise to a T-B-NK+ SCID phenotype. Thymic development is interrupted, and no T-lymphocytes are able to develop because of the intrinsic hematopoietic stem cell defect. In hypomorphic RAG deficiency, residual V(D)J function is permissive of some T-lymphocyte development, and so thymic development occurs, albeit in an aberrant fashion.

Increasing evidence demonstrates advancing restriction in the IGH and TRB repertoire associated with increased T- and B-lymphocyte clonality in patients with associated increasingly severe RAG deficiency, step-wise from normal through combined immunodeficiency with granulomas/autoimmunity, leaky SCID, and Omenn syndrome ⁴⁵ . A reduction in TCRβ diversity accompanied by distorted use of V, D, and J gene segments and restriction in CDR3 length diversity are more pronounced in patients with a more-severe clinical phenotype ⁴⁵ . This repertoire restriction is determined by the residual endonuclease activity conferred by the mutation in RAG1 ⁴⁶ or RAG2 ⁴⁷ , which permits some V(D)J recombination in less-severe mutations and permits the formation of a few T-lymphocyte clones which experience impaired positive and negative selection as they pass through the disrupted thymic structure.

However, more recent findings also directly implicate the effect of the RAG mutation on V(D)J recombination, preferentially conferring an autoreactive phenotype on the lymphocyte, leading to subsequent development of dysregulated immune responses, as well as the impact of the impaired central and peripheral tolerance mechanisms. Hypomorphic RAG mutations are more likely to lead to the development of an antigen receptor that is autoreactive as well as reduce the likelihood of deletion through central tolerance because of disrupted thymic architecture.

High-throughput sequencing of the T-lymphocyte receptor from patients with RAG mutations has demonstrated less junctional diversity and smaller CDR3 regions than seen in patients with Omenn syndrome caused by other gene mutations ⁴⁸ . During normal lymphocyte development, T-lymphocyte receptor and immunoglobulin gene rearrangements progress through a unique somatic recombination development. The TCRα locus employs a multistep recombination process, progressing from use of proximal TRAV elements to distal TRAV elements. This process is arrested by downregulation of RAG1/2 expression following positive selection of thymocytes. The persistent reduced recombinase activity over successive waves of TCRα rearrangement in patients with RAG defects manifests as a bias in TCRα use towards more proximal TRAV/TRAJ associations. These specific patterns can be detected using flow cytometric methods, which have been observed in patients with V(D)J recombination defects, including RAG deficiency ⁴⁹ .

Hypomorphic Rag murine models demonstrate impaired gene rearrangement at the Igh, Igk, and Trb loci, which limits antigen receptor diversity from a very early stage in thymocyte development ⁵⁰ . Hydrophobic residues within the T-lymphocyte receptor CDR3 region have been associated with the development of autoreactive T-lymphocytes ⁵¹ , and specific cysteine patterns in the same CDR3 region have also been shown to predict the development of autoreactivity ⁵² . In the conventional CD4+ T-lymphocytes of patients with combined immunodeficiency or atypical SCID, associated with hypomorphic RAG mutations, enhancement for hydrophobic amino acids associated with auto-reactivity and a reduction in those hydrophilic amino acids that limit autoimmunity have been identified in the CDR3 region ⁵³ . This may reveal an intrinsic bias favoring the production of auto-reactive T-lymphocytes as well as impaired negative selection due to impaired central tolerance, indicated by reduced expression of AIRE ⁴⁴ .

Additionally, there is a reduction in the number ^{43, 44, 50, 54} and function ^{53, 55} of regulatory T lymphocytes in these patients, which will have an impact on peripheral tolerance.

As well as a raised IgE, IgM and IgG autoantibodies have been detected in patients with hypomorphic RAG mutations, including those with Omenn syndrome, and especially those with autoimmune manifestations with combined immunodeficiency or granulomas, against a wide range of auto-antigens ⁵⁶ . Amongst these autoantibodies are neutralizing anti-cytokine antibodies directed against anti-IFN-α or anti-IFN-ω, which may be precipitated by environmental triggers such as viral infection ^{28, 57} . Murine models with hypomorphic Rag mutations produce high levels of low-affinity autoantibodies, associated with impaired receptor editing and an increase in serum B-lymphocyte-activating factor (BAFF) concentrations, also identified in patients with Omenn syndrome and leaky SCID, implicating B-lymphocytes in the autoreactivity found in these patients ⁵⁷ . BAFF is important in promoting B-lymphocyte survival. Reduced levels of BAFF receptor are expressed by anergic self-reactive B-lymphocytes compared to naïve B-lymphocytes ⁵⁸ , and in normal homeostasis, auto-reactive cells are therefore eliminated from the pool of circulating naïve B-lymphocytes. RAG-mutated patients and mouse models exhibit profound B-lymphocytopenia, and the higher circulating levels of BAFF enable the survival of immature auto-reactive B-lymphocytes ^{56, 59} . These patients have more class-switched immunoglobulin heavy chain transcripts and an increased rate of somatic hypermutation, indicating in vivo B lymphocyte activation ^{45, 57} .

Invariant NK T-lymphocyte populations protect against autoimmunity and graft-versus-host disease ^{60, 61} . Development requires thymic-dependent, RAG-initiated V(D)J recombination. These cells are absent in patients with Omenn syndrome, the lack of which may contribute to the autoimmune phenomena seen in these patients ⁶² .

Conventional NK cells are found in normal or sometimes increased numbers in patients with RAG mutations, regardless of the clinical presentation. Within the NK population, however, is a higher-than-normal proportion of immature (CD56 ^bright) NK cells, which exhibit both increased perforin content and enhanced degranulation activity ⁶³ . This heightened NK cell-mediated cytotoxicity may be responsible for the increased rate of graft rejection observed after hematopoietic stem cell transplantation (HSCT) in patients with RAG-deficient SCID ⁶⁴ , in contrast to patients with other NK+ SCID phenotypes, such as IL7Ra deficiency. An increased cytotoxic reaction has been observed also in NK lymphocytes from Rag ^–/– mice, although the NK cells display a mature and activated phenotype in contrast to patients ⁶⁵ .

The degree of residual protein function clearly plays a role in determining clinical phenotype, but it is not the sole determinant, because patients who share similar RAG mutations, which confer similar immunobiological effects, may present with different clinical phenotypes ⁶⁶ . Omenn syndrome can evolve from SCID over a period of weeks, as internal or external antigens, including viruses ¹⁴ , drive T-lymphocyte clonal expansion. Anti-cytokine antibodies, such as anti-IFN-α or anti-IFN-ω, seen in patients with RAG-mutated combined immunodeficiency or atypical SCID, may be produced as a result of viral infection in predisposed patients ^{57, 67} . The intestinal microbiota may also influence the development of autoimmunity. In murine models of RAG deficiency, inflammatory bowel disease mediated by intestinal CD4 T _H1/T _H17 T-lymphocytes has been ameliorated by manipulating the intestinal flora, resulting in the reduction of gut tropic T-lymphocytes and reduced IgE levels ⁶⁸ ( Table 2).

For the most-severe disease manifestations, including SCID, atypical SCID, and Omenn syndrome, HSCT is the standard of care. Initial management should be directed at the treatment of pre-existing infections, appropriate antimicrobial prophylaxis, and immunoglobulin replacement. Nutritional support with hyper alimentation or total parenteral nutrition is often required. Skin care is critical, and topical treatments should be utilized, including steroid ointments and topical tacrolimus, as well as topical anti-bacterial washes and emollients and barrier creams. Patients frequently experience super-added bacterial infection; Staphylococcus aureus is usually implicated. These infections should be treated aggressively. Calcineurin inhibitors and other systemic immunosuppression may improve the inflammation ⁶⁹ , which may affect any and all organs. Serotherapy may be required to remove auto-inflammatory cells, although immunosuppression in the presence of active infection can present specific challenges. Careful pre-transplant care is critical to a successful outcome following HSCT. In the modern era, survival from HSCT for primary immunodeficiencies is similar whether a matched sibling, matched unrelated, or mismatched T-lymphocyte-depleted donor is utilized. The outcome of HSCT for RAG deficiency (characterized as T-B- SCID) has been significantly worse than other forms of SCID ⁷⁰ , but a recent report has shown similar survival rates for RAG-SCID as for other genetically defined T-B+ SCID phenotypes ⁷¹ . Thymic and bone marrow niches are occupied by developmentally arrested pre-cursors in RAG-deficient patients, and so long-term immune reconstitution is likely to be achieved only after the use of a conditioning regimen ⁷² , preferentially with serotherapy which targets NK cells. Graft failure is common in RAG deficiency ^{64, 71} , perhaps partly mediated by highly reactive NK cells which exhibit enhanced degranulation and increased amounts of perforin ⁶³ . The best results are achieved if patients are infection-free at the time of transplantation ^{71, 73} , most likely accomplished if they are detected by newborn screening ⁷⁴ .

Genetic therapy using gene addition is likely to be the next therapeutic advance in treating these patients. Autologous stem cells are used, removing the risk of graft-versus-host disease and rejection. Gene therapy for X-linked SCID and for adenosine deaminase-deficient SCID has been established, with a licensed vector for adenosine deaminase-deficient SCID available in Europe. Survival results are excellent, and the lentiviral vectors have a better safety profile than gammaretroviral vectors ^{75, 76} . Preclinical lentiviral vector gene therapy studies for RAG1 and RAG2 deficiency using animal models have shown some success ^{77, 78} to date without the risks of oncogenesis described when older retroviral vectors were used ⁷⁹ ; clinical trials using lentiviral vectors to treat patients are now in progress ⁸⁰ . The very best outcomes may be possible when patients are treated infection-free as newborns using a gene therapy protocol with chemotherapy-free antibody-based conditioning regimens ^{81, 82} .

It is now clear that hypomorphic mutations in RAG genes can result in protean manifestations of immunodeficiency, immunodysregulation, or autoimmunity, even manifesting in late adolescence or early adulthood. New understandings of the biological effects of these mutations, particularly on the development of T- and B-lymphocyte receptors, help explain these clinical presentations. For the more-severe manifestations, emerging gene therapy technologies may offer novel therapy options.