RAG-1-deficient mice have no mature B and T lymphocytes.

The V(D)J recombination activation gene RAG-1 was isolated on the basis of its ability to activate V(D)J recombination on an artificial substrate in fibroblasts. This property and the expression pattern in tissues and cell lines indicate that RAG-1 either activates or catalyzes the V(D)J recombination reaction of immunoglobulin and T cell receptor genes. We here describe the introduction of a mutation in RAG-1 into the germline of mice via gene targeting in embryonic stem cells. RAG-1-deficient mice have small lymphoid organs that do not contain mature B and T lymphocytes. The arrest of B and T cell differentiation occurs at an early stage and correlates with the inability to perform V(D)J recombination. The immune system of the RAG-1 mutant mice can be described as that of nonleaky scid mice. Although RAG-1 expression has been reported in the central nervous system of the mouse, no obvious neuroanatomical or behavioral abnormalities have been found in the RAG-1-deficient mice.     

Irradiation promotes V(D)J joining and RAG-dependent neoplastic transformation in SCID T-cell precursors

Defects in the nonhomologous end-joining (NHEJ) pathway of double-stranded DNA break repair severely impair V(D)J joining and selectively predispose mice to the development of lymphoid neoplasia. This connection was first noted in mice with the severe combined immune deficient (SCID) mutation in the DNA-dependent protein kinase (DNA-PK). SCID mice spontaneously develop thymic lymphoma with low incidence and long latency. However, we and others showed that low-dose irradiation of SCID mice dramatically increases the frequency and decreases the latency of thymic lymphomagenesis, but irradiation does not promote the development of other tumors. We have used this model to explore the mechanistic basis by which defects in NHEJ confer selective and profound susceptibility to lymphoid oncogenesis. Here, we show that radiation quantitatively and qualitatively improves V(D)J joining in SCID cells, in the absence of T-cell receptor-mediated cellular selection. Furthermore, we show that the lymphocyte-specific endonuclease encoded by the recombinase-activating genes (RAG-1 and RAG-2) is required for radiation-induced thymic lymphomagenesis in SCID mice. Collectively, these data suggest that irradiation induces a DNA-PK-independent NHEJ pathway that facilitates V(D)J joining, but also promotes oncogenic misjoining of RAG-1/2-induced breaks in SCID T-cell precursors.     

T and B cell recovery in arthritis adoptively transferred to SCID mice: antigen-specific activation is required for restoration of autopathogenic CD4+ Th1 cells in a syngeneic system

T cell homeostasis is a physiological function of the immune system that maintains a balance in the numbers and ratios of T cells at the periphery. A self-MHC/self-peptide ligand can induce weak (covert) signals via the TCR, thus providing an extended lifespan for naive T cells. A similar mechanism is responsible for the restoration of immune homeostasis in severe lymphopenic conditions such as those following irradiation or chemotherapy, or upon transfer of lymphocytes to nu/nu or SCID mice. To date, the genetic backgrounds of donor and recipient SCID mice were unmatched in all autoimmune arthritis transfer experiments, and the recovery of lymphoid cells in the host has not been followed. In this study, we present the adoptive transfer of proteoglycan (PG)-induced arthritis using unseparated and T or B cell-depleted lymphocytes from arthritic BALB/c donors to genetically matched syngeneic SCID recipient mice. We demonstrate that selectively recovered lymphoid subsets determine the clinical and immunological status of the recipient. We found that when T cells were depleted (>98% depleted), B cells did not produce PG-specific anti-mouse (auto) Abs unless SCID mice received a second Ag (PG) injection, which promoted the recovery of Ag-specific CD4(+) Th1 cells. Reciprocally, as a result of B cell recovery, high levels of serum anti-PG Abs were found in SCID mice that received B cell-depleted (>99% depleted) T lymphocytes. Our results indicate a selective and highly effective cooperation between CD4(+) T cells and B lymphocytes that is required for the restoration of pathological homeostasis and development of autoimmune arthritis in SCID mice.     

Generation of normal lymphocyte populations by Rb-deficient embryonic stem cells

BACKGROUND: Mice homozygous for a loss-of-function mutation of the recombination-activating gene-2 (RAG 2), which is required for the rearrangement of antigen receptor genes, do not produce mature B and T lymphocytes. But chimeric mice that result from injection of normal embryonic stem (ES) cells into blastocysts from RAG2-deficient mice develop normal mature lymphocyte populations, all of which are derived from the injected ES cells; we have called this process RAG2-deficient blastocyst complementation. Using ES cells with homozygous mutations, RAG-2-deficient blastocyst complementation could provide a physiological assay with which to determine the potential role of almost any gene in the development and/or function of lymphocytes. To test the general utility of this system, we have used it to test the differentiation-potential of ES cells that harbor homozygous loss-of function mutations of their retinoblastoma susceptibility (Rb) gene loci. We chose Rb for this analysis because of its widespread function in the control of the cell cycle and cell differentiation, the adverse effect of homozygous germline mutations of Rb on hematopoiesis in fetal liver, and the embryonic lethality that results when the homozygous Rb mutation is introduced into the germline. RESULTS: Homozygous Rb mutant ES cells can develop into phenotypically normal, mature B and T lymphocytes in the RAG-2-deficient background. Strikingly, Rb-deficient B and T cells do not have major defects in either activation or function. CONCLUSION: We have demonstrated the efficacy of the RAG-2-deficient blastocyst complementation system for evaluating the role of critical genes in lymphocyte development. Our results indicate that Rb expression is not intrinsically required for B-cell or T-cell function, despite the normally high levels of Rb expressed in lymphoid cells.     

Definition of a core region of RAG-2 that is functional in V(D)J recombination.

The products of the RAG-1 and RAG-2 genes cooperate to allow V(D)J recombination in lymphoid and non-lymphoid cells. As one step toward understanding the role of RAG-2, we have constructed mutated RAG-2 genes and examined their ability to support recombination of plasmid substrates in a fibroblast cell line. The mutations define essential and dispensable parts of the RAG-2 gene. Mutations in the N-terminal part eliminate almost all activity. In the central region of the protein, some but not all local alterations still allow recombination. On the other hand, proteins with large deletions from the C-terminal end, including one truncated by 25%, still retain activity, even though this part of the protein is highly conserved between species. Similar results were obtained with substrates that retain either a signal joint or a coding joint, or perform an inversion. Thus all basic features of V(D)J joining are retained in a RAG-2 protein with only the first 75% of the sequence.     

Selective expression of RAG-2 in chicken B cells undergoing immunoglobulin gene conversion

Chickens create their immunoglobulin (Ig) repertoires during B cell development in the bursa of Fabricius by intrachromosomal gene conversion. Recent evidence has suggested that Ig gene conversion may involve cis-acting DNA elements related to those involved in V(D)J recombination. Therefore, we have examined the potential role of the V(D)J recombination activating genes, RAG-1 and RAG-2, in regulating chicken Ig gene conversion. In contrast to the coexpression of RAG-1 and RAG-2 observed in mammalian B cells that undergo V(D)J recombination, chicken B cells isolated from the bursa of Fabricius express high levels of the RAG-2 mRNA but do not express RAG-1 mRNA. The developmental and phenotypic characteristics of the bursal lymphocytes and chicken B cell lines that express RAG-2 mRNA demonstrate that selective RAG-2 expression occurs specifically in B cells undergoing Ig diversification by gene conversion. These data suggest that RAG-2 plays a fundamental role in Ig-specific gene conversion.     

Lymphoid bone marrow cultures can reconstitute heterogeneous B and T cell-dependent responses in severe combined immunodeficient mice

Long-term lymphoid bone marrow cultures (LBMC) produce B lymphocytes and their precursors for several months in vitro. To assess their differentiative potential and determine their capacity to function as immune effectors, cells from the cultures were transplanted into mice with severe combined immune deficiency disease (SCID). SCID mice are deficient in T and B lymphocytes and are serum immunoglobulin (Ig) negative, but grafts of normal lymphoid precursors can expand and differentiate in them, thereby restoring immunocompetence. The results of these studies indicate that cells from LBMC are able to reconstitute splenic B lymphocytes in the SCID mice. Upon in vivo transfer, LBMC cells secreted Ig that displayed isotype distribution and a pattern of heterogeneity comparable with normal BALB/c mice, as determined by two-dimensional gel electrophoresis. The transplanted LBMC cells were functional, because reconstituted mice could respond to immunization with the T-independent antigen TNP-Ficoll. The results also indicate that cultured cells could reconstitute T cell activity in SCID mice. Splenocytes from approximately one-third of the recipients could generate a cytotoxic response to alloantigens after 5 days of sensitization in a mixed lymphocyte culture, and all reconstituted SCID mice could respond to immunization with the T cell-dependent antigen TNP-BSA. These results demonstrate that B cells, as well as T cell activity, are present in LBMC-reconstituted SCID mice, and show that LBMC cells have the capacity to mediate an immune response.     

Cutting edge: ectopic expression of the chemokine TCA4/SLC is sufficient to trigger lymphoid neogenesis

To test whether accumulation of naive lymphocytes is sufficient to trigger lymphoid development, we generated mice with islet expression of the chemokine TCA4/SLC. This chemokine is specific for naive lymphocytes and mature dendritic cells (DC) which express the CCR7 receptor. Islets initially developed accumulations of T cells with DC, with scattered B cells at the perimeter. These infiltrates consolidated into organized lymphoid tissue, with high endothelial venules and stromal reticulum. Infiltrate lymphocytes showed a naive CD44low CD25- CD69- phenotype, though half were CD62L negative. When backcrossed to RAG-1 knockout, DC were not recruited. Interestingly, islet lymphoid tissue developed in backcrosses to Ikaros knockout mice despite the absence of normal peripheral nodes. Our results indicate that TCA4/SLC can induce the development and organization of lymphoid tissue through diffential recruitment of T and B lymphocytes and secondary effects on stromal cell development.     

ATM prevents the persistence and propagation of chromosome breaks in lymphocytes

DNA double-strand breaks (DSBs) induce a signal transmitted by the ataxia-telangiectasia mutated (ATM) kinase, which suppresses illegitimate joining of DSBs and activates cell-cycle checkpoints. Here we show that a significant fraction of mature ATM-deficient lymphocytes contain telomere-deleted ends produced by failed end joining during V(D)J recombination. These RAG-1/2 endonuclease-dependent, terminally deleted chromosomes persist in peripheral lymphocytes for at least 2 weeks in vivo and are stable over several generations in vitro. Restoration of ATM kinase activity in mature lymphocytes that have transiently lost ATM function leads to loss of cells with terminally deleted chromosomes. Thus, maintenance of genomic stability in lymphocytes requires faithful end joining as well a checkpoint that prevents the long-term persistence and transmission of DSBs. Silencing this checkpoint permits DNA ends produced by V(D)J recombination in a lymphoid precursor to serve as substrates for translocations with chromosomes subsequently damaged by other means in mature cells.     

Rag-1 mutations associated with B-cell-negative scid dissociate the nicking and transesterification steps of V(D)J recombination

Some patients with B-cell-negative severe combined immune deficiency (SCID) carry mutations in RAG-1 or RAG-2 that impair V(D)J recombination. Two recessive RAG-1 mutations responsible for B-cell-negative SCID, R621H and E719K, impair V(D)J recombination without affecting formation of single-site recombination signal sequence complexes, specific DNA contacts, or perturbation of DNA structure at the heptamer-coding junction. The E719K mutation impairs DNA cleavage by the RAG complex, with a greater effect on nicking than on transesterification; a conservative glutamine substitution exhibits a similar effect. When cysteine is substituted for E719, RAG-1 activity is enhanced in Mn(2+) but remains impaired in Mg(2+), suggesting an interaction between this residue and an essential metal ion. The R621H mutation partially impairs nicking, with little effect on transesterification. The residual nicking activity of the R621H mutant is reduced at least 10-fold upon a change from pH 7.0 to pH 8.4. Site-specific nicking is severely impaired by an alanine substitution at R621 but is spared by substitution with lysine. These observations are consistent with involvement of a positively charged residue at position 621 in the nicking step of the RAG-mediated cleavage reaction. Our data provide a mechanistic explanation for one form of hereditary SCID. Moreover, while RAG-1 is directly involved in catalysis of both nicking and transesterification, our observations indicate that these two steps have distinct catalytic requirements.     

Cell cycle-dependent accumulation in vivo of transposition-competent complexes between recombination signal ends and full-length RAG proteins

V(D)J recombination is initiated by a specialized transposase consisting of RAG-1 and RAG-2. Because full-length RAG proteins are insoluble under physiologic conditions, most previous analyses of RAG activity in vitro have used truncated core RAG-1 and RAG-2 fragments. These studies identified an intermediate in V(D)J recombination, the signal end complex (SEC), in which core RAG proteins remain associated with recombination signal sequences at the cleaved signal ends. From transfected cells expressing affinity-tagged RAG proteins, we have isolated in vivo assembled SECs containing full-length RAG proteins and cleaved recombination substrates. SEC formation in vivo did not require the repair proteins DNA-dependent protein kinase, Ku80, or XRCC4. In the presence of full-length RAG-2, SEC formation in vivo was cell cycle-regulated and restricted to the G(0)/G(1) phases. In contrast, complexes accumulated throughout cell cycle in cells expressing a RAG-2 CDK2 phosphorylation site mutant. Both core and full-length SECs supported transposition in vitro with similar efficiencies. Intracellular SECs, which are likely to persist in the absence of coding ends, represent potential donors whose transposition is not suppressed by the non-core regions of the RAG proteins.     

The V(D)J recombination activating gene, RAG-1

The RAG-1 (recombination activating gene-1) genomic locus, which activates V(D)J recombination when introduced into NIH 3T3 fibroblasts, was isolated by serial genomic transfections of oligonucleotide-tagged DNA. A genomic walk spanning 55 kb yielded a RAG-1 genomic probe that detects a single 6.6-7.0 kb mRNA species in transfectants and pre-B and pre-T cells. RAG-1 genomic and cDNA clones were biologically active when introduced into NIH 3T3 cells. Nucleotide sequencing of human and mouse RAG-1 cDNA clones predicts 119 kd proteins of 1043 and 1040 amino acids, respectively, with 90% sequence identity. RAG-1 has been conserved between species that carry out V(D)J recombination, and its pattern of expression correlates exactly with the pattern of expression of V(D)J recombinase activity. RAG-1 may activate V(D)J recombination indirectly, or it may encode the V(D)J recombinase itself.     

Gamma-irradiation directly affects the formation of coding joints in SCID cell lines.

SCID mice have a defect in the catalytic subunit of the DNA-dependent protein kinase, causing increased sensitivity to ionizing radiation in all tissues and severely limiting the development of B and T cell lineages. SCID T and B cell precursors are unable to undergo normal V(D)J recombination: coding joint and signal joint products are less frequently formed and often will exhibit abnormal structural features. Paradoxically, irradiation of newborn SCID mice effects a limited rescue of T cell development. It is not known whether irradiation has a direct impact on the process of V(D)J joining, or whether irradiation of the thymus allows the outgrowth of rare recombinants. To investigate this issue, we sought to demonstrate an irradiation effect ex vivo. Here we have been able to reproducibly detect low-frequency coding joint products with V(D)J recombination reporter plasmids introduced into SCID cell lines. Exposure of B and T lineage cells to 100 cGy of gamma irradiation made no significant difference with respect to the number of coding joint and signal joint recombination products. However, in the absence of irradiation, the coding joints produced in SCID cells had high levels of P nucleotide insertion. With irradiation, markedly fewer P insertions were seen. The effect on coding joint structure is evident in a transient assay, in cultured cells, establishing that irradiation has an immediate impact on the process of V(D)J recombination. A specific proposal for how the DNA-dependent protein kinase catalytic subunit influences the opening of hairpin DNA intermediates during coding joint formation in V(D)J recombination is presented.     

T-cell receptor alpha locus V(D)J recombination by-products are abundant in thymocytes and mature T cells.

In addition to the assembled coding regions of immunoglobulin and T-cell receptor (TCR) genes, the V(D)J recombination reaction can in principle generate three types of by-products in normal developing lymphocytes: broken DNA molecules that terminate in a recombination signal sequence or a coding region (termed signal or coding end molecules, respectively) and DNA molecules containing fused recombination signal sequences (termed reciprocal products). Using a quantitative Southern blot analysis of the murine TCR alpha locus, we demonstrate that substantial amounts of signal end molecules and reciprocal products, but not coding end molecules, exist in thymocytes, while peripheral T cells contain substantial amounts of reciprocal products. At the 5' end of the J alpha locus, 20% of thymus DNA exists as signal end molecules. An additional 30 to 40% of the TCR alpha/delta locus exists as remarkably stable reciprocal products throughout T-cell development, with the consequence that the TCR C delta region is substantially retained in alpha beta committed T cells. The disappearance of the broken DNA molecules occurs in the same developmental transition as termination of expression of the recombination activating genes, RAG-1 and RAG-2. These findings raise important questions concerning the mechanism of V(D)J recombination and the maintenance of genome integrity during lymphoid development.     

Identification of multiple isolated lymphoid follicles on the antimesenteric wall of the mouse small intestine.

We have revealed that 100-200 clusters, filled with closely packed lymphocytes, can be found throughout the length of the antimesenteric wall of the mouse small intestine. They are composed of a large B cell area, including a germinal center, and epithelia overlying the clusters contain M cells. A large fraction of B cells displays B220+ CD19+ CD23+ IgM(low)IgD(high)CD5(-)Mac-1(-) phenotype, and the composition of IgA+ B cells is smaller but substantial. To our knowledge, these clusters are the first identification of isolated lymphoid follicles (ILF) in mouse small intestine. ILF can be first detected at 7 (BALB/c mice) and 25 (C57BL/6 mice) days after birth, and lymphoid clusters equivalent in terms of cellular mass to ILF are present in germfree, athymic nude, RAG-2(-/-), TCR-beta(-/-), and Ig mu-chain mutant (mu(-/-)) mice, although c-kit+ cells outnumber B220+ cells in germfree and athymic nude mice, and most lymphoid residents are c-kit+ B220(-) in RAG-2(-/-), TCR-beta(-/-), and mu(-/-) mice. ILF develop normally in the progeny of transplacentally manipulated Peyer's patch (PP)-deficient mice, and decreased numbers of conspicuously atrophied ILF are present in IL-7Ralpha(-/-) PP(null) mice. Neither ILF nor PP are detectable in lymphotoxin alpha(-/-) and aly/aly mice that retain well-developed cryptopatches (CP) and thymus-independent subsets of intraepithelial T cells, whereas ILF, PP, CP, and thymus-independent subsets of intraepithelial T cells disappear from common cytokine receptor gamma-chain mutant mice. These findings indicate that ILF, PP, and CP constitute three distinct organized gut-associated lymphoid tissues that reside in the lamina propria of the mouse small intestine.     

CD4+CD8- thymocytes from neonatal mice induce IgM production in SCID mice.

Defective recombination of both the TCR and Ig genes results in the absence of mature lymphocytes in mice with the scid mutation. We have shown previously that the transfer of neonatal, but not adult, thymocytes results in high levels of Ig production in 100% of C.B-17-scid (SCID) mice, in contrast to the 10 to 25% of SCID mice spontaneously producing low levels of oligoclonal Ig. In this report we demonstrate that neonatal CD4+8- thymocytes were able to induce this response; the CD4+8+ and CD4-8+ subpopulations were totally inactive and CD4-8- T cells had only limited activity several weeks after transfer. The stimulation of IgM production in SCID mice was detectable by 1 wk posttransfer of CD4+8- thymocytes or splenic T cells, and could be achieved with as few as 300 cells. The ability of neonatal CD4+8- thymocytes to induce Ig diminished gradually to insignificant levels at 3 wk postbirth; this loss of function was not associated with differential survival of neonatal T cells. Neonatal CD4+8- thymocytes from C.B-17 and other H-2d strains rescued Ig production, whereas cells from H-2b, H-2a, and H-2k strains were much less effective. These results suggest that a CD4+8- subpopulation found in both neonatal thymus and peripheral lymphoid tissues is able to induce the expansion or differentiation of the small numbers of functional B lymphocytes in SCID mice, and that the inducing T cell disappears shortly after birth, perhaps during the acquisition of self-tolerance.