The scid mutation in mice causes a general defect in DNA repair.

Mice homozygous for the scid mutation on chromosome 16 have a severe combined immune deficiency as a result of their inability to correctly rearrange their immunoglobulin and T-cell receptor genes. In scid mice, when precursors for B and T lymphocytes reach the stage of development requiring expression of these surface receptors, a defective recombinase system aberrantly cuts and rejoins the receptor gene segments greatly reducing the efficiency of producing functional receptors. As a result, most scid mice have no detectable B or T lymphocytes. We have demonstrated that the scid defect is not specific to lymphocyte development. Myeloid cells and fibroblasts from scid mice show a marked increase in sensitivity to ionizing radiation, indicating that the scid mutation leads to an inability to repair DNA damage induced by ionizing radiation as well as interfering with rearrangement of the immunoglobulin and T-cell receptor genes.

Use of scid mice to identify and quantitate lymphoid-restricted stem cells in long-term bone marrow cultures.

Mice homozygous for an autosomal recessive scid (severe combined immune deficiency) mutation on chromosome 16 exhibit a defect that specifically impairs lymphoid differentiation but not myelopoiesis. Consequently such mice are deficient in both humoral and cell-mediated immune functions. Despite their defect, scid mice survive under pathogen-free conditions and are fertile. The mutation does not impair the hematopoietic microenvironment necessary for lymphoid differentiation, since these mice can be cured with grafts of normal bone marrow (BM) or cells from long-term BM cultures (LTBMC); however, reconstitution requires sublethal (400 cGy) irradiation of recipients. Engraftment with cells from LTBMC gave near-normal levels of colony-forming B cells (CFU-B) in spleen and BM of the recipients by 6 weeks postgrafting. Since LTBMC are devoid of all mature B and pre-B cells but contain stem cells that restore lymphoid function in scid mice, we used a limiting-dilution assay to characterize and enumerate the number of stem cells in LTBMC capable of restoring lymphoid function. Curing was determined by the CFU-B-cell assay, since CFU-B are not detectable in normal scid mice. The results indicate that fewer cells from LTBMC than from fresh BM are required to obtain lymphoid reconstitution. As few as 10(3) LTBMC cells can repopulate significant B- and T-cell function in scid recipients. From these results we conclude that scid mice can be used as recipients to quantify lymphoid-restricted stem cells and that there is a functional separation of lymphoid- and myeloid-restricted stem cells in LTBMC with an enrichment for lymphoid-restricted stem cells in these cultures.

Murine lymphocytes with natural killer activity express CTL-derived serine protease genes.

Serine protease genes (C11, B10 and HF) derived from activated cytolytic T lymphocytes have been shown to be important in CTL-mediated cytotoxicity. In this study, we examined the expression of these genes in fresh natural killer (NK) cells from severe combined immunodeficiency (SCID) and athymic nude mice, as well as in T-cell lines with NK activity. All of these serine protease genes were expressed in NK cells freshly isolated from SCID and athymic nude mice. In addition, all lines showed similar strong levels of expression of C11 and B10 genes, but not the HF gene. However, levels of expression in the T-cell lines did not correlate with levels of NK-like cytotoxicity. These results suggest that C11, B10 and HF serine protease genes are necessary but not sufficient for NK-like cytotoxicity.

Isolation of scid pre-B cells that rearrange kappa light chain genes: formation of normal signal and abnormal coding joins.

Consistent with an ordered immunoglobulin (Ig) gene assembly process during precursor (pre-) B cell differentiation, we find that most Abelson murine leukemia virus (A-MuLV)-transformed pre-B cells derived from scid (severe combined immune deficient) mice actively form aberrant rearrangements of their Ig heavy chain locus but do not rearrange endogenous kappa light chain variable region gene segments. However, we have identified several scid A-MuLV transformants that transcribe the germline Ig kappa light chain constant region and actively rearrange the kappa variable region gene locus. In one case progression to the stage of kappa light chain gene rearrangement did not require expression of Ig mu heavy chains; furthermore, this progression could not be efficiently induced following expression of mu heavy chains from an introduced vector. As observed in pre-B cell lines from normal mice, attempted V kappa-to-J kappa rearrangements in scid transformants occur by inversion at least as frequently as by deletion. The inverted rearrangements result in retention of both products of the recombination event in the chromosome, thus allowing their examination. scid kappa coding sequence joins are aberrant and analogous in structure to previously described scid heavy chain coding joins. In contrast, the recognition signals that flank involved coding segments frequently are joined precisely back-to-back in normal fashion. The scid VDJ recombinase defect therefore does not significantly impair recognition of, site-specific cutting at, or juxtaposition and appropriate ligation of signal sequences. Our finding that the scid defect prevents formation of correct coding but not signal joins distinguishes these events mechanistically.

The scid mouse as a model to identify and quantify myeloid and lymphoid stem cells.

Scid mice are excellent recipients for studying the characteristics of stem cells. Sublethal irradiation not only enhances engraftment of stem cells but enables one to graft limiting numbers of cells without compromising the survival of the recipient. This enables one to estimate the frequency of stem cells by limiting dilution analysis. Compared to fresh bone marrow, LTBMC are slightly enriched for stem cells capable of reconstituting lymphoid function in scid recipients. The stem cells have self-renewal ability since bone marrow from cured primary scid recipients can cure secondary recipients. Our results indicate that lymphoid reconstitution following engraftment with LTBMC occurs from a lymphoid-restricted stem cell; similar restricted stem cells also exist in normal bone marrow.

The effect of the scid mutation on mechanism and control of immunoglobulin heavy and light chain gene rearrangement.

Most Abelson murine leukemia virus (A-MuLV)-transformed cell lines derived from scid (severe combined immune deficient) mice actively rearrange their endogenous immunoglobulin (Ig) heavy (H), but not light (L) chain variable region genes. Such cell lines express germline VH segments and other RNA transcripts that are characteristically produced by early precursor (pre)-B lymphocytes, but do not express high levels of transcripts from the germline kappa (k) constant region (C kappa) locus. However, we have derived scid A-MuLV transformants that express germline C kappa transcripts and attempt kappa gene assembly. In one case kappa gene expression and rearrangement occurred in the absence of mu H chain expression, and in another was not induced efficiently by introduction of a mu-expression vector. Although the vast majority of scid H and L chain coding sequence joins are grossly aberrant, scid A-MuLV transformants can form normal coding joints at a very low frequency. In contrast, these cells form generally normal signal sequence joins at an approximately normal efficiency. Thus, these findings mechanistically distinguish coding and signal join formation. Subcloning analyses suggest that scid A-MuLV transformants that do not attempt chromosomal coding sequence joining may have a relative survival advantage, and therefore that these events may often result in unrepaired chromosomal breakage and cell death.

The scid defect affects the final step of the immunoglobulin VDJ recombinase mechanism.

Abelson murine leukemia virus-transformed precursor B lymphocytes from scid (severe combined immunodeficient) mice, like A-MuLV transformants from normal mice, actively rearrange segments of their Ig heavy chain variable region gene locus during growth in culture. Targeting of recombination to appropriate segments appears normal in these lines as evidenced by initial rearrangement of sequences from within the D and JH locus to form aberrant "DJH" rearrangements and secondary rearrangement of sequences from within the VH locus to the aberrant "DJH" intermediates. A detailed analysis of the joints in these rearrangements indicates that the VDJ recombinase in scid pre-B cells can correctly recognize heptamernonamer signal sequences and perform precise endonucleolytic scissions at these sequences. We propose that the scid defect involves the inability of scid precursor lymphocytes to join correctly the cleaved ends of the coding strands of variable region gene segments.

Early B-cell precursors in scid mice: normal numbers of cells transformable with Abelson murine leukemia virus (A-MuLV).

scid mice lack detectable B and T lymphocytes; there are no typical pre-B cells as defined by c mu and surface markers in their bone marrow and their thymus contains only 1% of the normal number of cells. In these characters scid mice seem to lack lymphoid stem cells. However, some mice have detectable serum immunoglobulin and others develop thymomas; both observations indicate that the block in lymphoid development is not absolute. To determine whether scid mice have any B-cell precursors, we looked for pre-B cells by their ability to be transformed by Abelson murine leukemia virus (A-MuLV). Surprisingly, scid mice contain as many B-cell precursors transformable with A-MuLV as normal control mice. Cell-surface markers specific for pre-B and B cells were detected on the A-MuLV-transformed bone marrow cells of both scid and normal mice, indicating that the A-MuLV-transformed cells belong to the B lineage. Interestingly, the same surface markers were undetectable on nontransformed scid bone marrow cells. We conclude from these results that scid mice have normal numbers of early B-cell precursors but that their differentiation into functional B cells is severely impaired.

An expanded population of natural killer cells in mice with severe combined immunodeficiency (SCID) lack rearrangement and expression of T cell receptor genes.

Mice with severe combined immunodeficiency syndrome (SCID) exhibit an impairment in both T and B cell maturation, whereas myelopoiesis remains unaffected. We report here that spleens from SCID mice have undergone phenotypic expansion of cells bearing the NK-2 and asialo GM1 markers (70-80%) characteristic of NK cells and this expansion is accompanied by a 3-4-fold enrichment in NK cytolytic activity over their normal C.B-17 littermates. Furthermore, the NK cells from SCID mice do not rearrange or express T cell receptor alpha or beta genes, or a third T cell rearranging gene, gamma. These findings suggest that (a) T cell receptors are not necessary for NK-mediated cytolysis, and (b) either NK cells constitute an entirely distinct lineage or NK cell function is acquired in pre-T cells prior to the expression of T cell receptor genes.

Full reconstitution of the immune deficiency in scid mice with normal stem cells requires low-dose irradiation of the recipients.

Mice homozygous for an autosomal recessive mutation for the scid gene exhibit a defect that specifically impairs lymphoid differentiation but not myelopoiesis. Such mice can be cured of their lymphoid deficiency by grafts with normal bone marrow, although full reconstitution of lymphoid function is seldom obtained. Long-term bone marrow cultures (LTBMC) are devoid of all mature B and pre-B cells but contain lymphoid stem cells. We therefore reconstituted scid mice with LTBMC cells to study the kinetics of B lymphocyte reconstitution in normal and irradiated (4 Gy) scid recipients and in irradiated (9.5 Gy) co-isogenic C.B-17 mice. Detectable colony-forming B cells rapidly increased in the spleen and bone marrow of irradiated C.B-17 and irradiated scid recipients, reaching normal levels between 4 and 6 wk post-grafting. Unirradiated scid recipients showed limited reconstitution in spleen and very poor reconstitution in bone marrow. Unirradiated scid recipients also had relatively few surface Ig+ cells in spleen or bone marrow, whereas both groups of irradiated recipients had normal numbers between 4 and 6 wk post-reconstitution. Normal levels of cytotoxic T cell activity by 8 wk after reconstitution were observed only in the irradiated C.B-17 and irradiated scid recipients. Analysis of mice reconstituted with cells from LTBMC indicates that these cultures contain lymphoid stem cells with significant proliferative and self-renewal potential, and that full reconstitution of lymphoid function requires prior irradiation of the scid recipient.

Regulation of bone marrow lymphocyte production: IV. Altered kinetic steady state of lymphocyte production after chronic changes in exogenous stimuli.

A transient increase in B lymphocyte production in mouse bone marrow has previously been shown to follow a single administration of various exogenous agents. The effect of sustained changes in exogenous stimuli on the level of bone marrow B lymphocyte production has now been studied. In mice given multiple injections of sheep red blood cells (SRBC) for four weeks the production of bone marrow lymphocytes was augmented, as indicated by increased numbers of cytoplasmic mu-chain-bearing pre-B cells and of surface mu-chain-bearing B lymphocytes, as well as increased rates of pre-B cell proliferation and small lymphocyte turnover. In an attempt to reduce potential external stimuli, mice were raised on an elemental diet. When compared to conventionally reared mice, however, they showed little difference in bone marrow small lymphocyte production and an identical pre-B cell proliferation rate. In addition, the small lymphocyte production rate in the thymus was not consistently altered either in SRBC-treated mice or elemental diet-fed mice, whereas small lymphocyte renewal in the spleen showed changes reflecting those in the bone marrow. The results demonstrate that a chronic increase in exposure to extrinsic agents can produce a long-term elevation of the population size and production rate of bone marrow B lineage cells. This suggests that the level of the kinetic steady state of primary B lymphocyte production normally observed in the bone marrow may reflect the level of exposure to potential stimulants in the external environment.

In vivo regulation of B lymphocyte production in the bone marrow: effects and mechanism of action of exogenous stimuli on pre-B cell proliferation and lymphocyte turnover.

The present studies demonstrate that a single administration of an extrinsic agent (SRBC) can stimulate increased production of B lymphocytes in mouse bone marrow as revealed by 2 in vivo assays which quantitate pre-B cell proliferation and small lymphocyte renewal, respectively. The mechanisms mediating this stimulatory effect are sensitive to silica in vivo and require the presence of the spleen. Early events are both silica-sensitive and spleen-dependent, while a subsequent stage appears still to be spleen-dependent but not silica-sensitive. Sustained exogenous stimulation by multiple SRBC injections for 4 wk in young mice produces an expanded population size and increased production of pre-B cells and B lymphocytes in the bone marrow, apparently an elevated kinetic steady state of B lymphocyte production. (Formula: see text). As depicted schematically in Figure 1, the results suggest that the magnitude of bone marrow B lymphocyte production in vivo may reflect a basal level, putatively regulated by microenvironmental and other endogenous factors, which is amplified by exogenous environmental stimuli mediated by the action of macrophages located in the spleen. Further questions about such an environmental amplification (Fig. 1) concern the nature of later events in the spleen, the identity of putative stimulatory factors or cells circulating from the spleen to the bone marrow, the receptive target cell stages in the bone marrow and the consequences of this process with respect to the size and diversity of B lymphocyte clones and of primary humoral immune responses in vivo.

Phagocytosis in the spleen of the sunfish Lepomis spp.

A histological investigation of the filtering function of the spleen of the sunfish Lepomis spp. was conducted by light, scanning, and transmission electron microscopy. The parenchyma of the organ is predominantly red pulp, a system of splenic cords and sinuses. The white pulp consists of loose lymphoid tissue which forms a cuff around the pulp arteries. Filtering of particulate matter from the blood occurs in the red pulp by phagocytes of the pulp cords and ellipsoids (periarterial macrophage sheaths). The ellipsoids are pale-staining cuffs of macrophages and reticular cells in a framework of reticular fibres surrounding the arterial capillaries. Destruction of effete blood cells (especially erythrocytes) is confined to the pigment nodules; particulate matter is not taken up by the nodules. These yellow-brown bodies are dispersed throughout the red pulp and are bounded by a reticular capsule. They contain masses of phagocytes and have the appearance of a morula. They are associated with blood vessels and are surrounded by sinusoids. Prussian Blue stain shows the presence of haemosiderin within their phagocytes. The phagocytes of the pigment nodules are filled with inclusions such as residual bodies, siderosomes, and fragments of erythrocytes. The early filtering of particulate matter by the phagocytes of the pulp cords and ellipsoids may allow for a more efficient phagocytosis of erythrocytes by the pigment nodules, followed by storage and reutilization of iron-containing compounds uncontaminated by other phagocytosed material.

Regulation of bone marrow lymphocyte production. III. Increased production of B and non-B lymphocytes after administering systemic antigens.

To examine the influence of exogenous stimuli on the genesis of lymphocytes in mouse bone marrow, the production rate and subsets of marrow lymphocytes were examined after a systemic injection of sheep red blood cells (SRBC). Radioautographic analysis after either pulse labeling or infusion of [3H]thymidine revealed a pronounced increase in the number of newly formed small lymphocytes appearing in the marrow, maximal 4-5 days after SRBC injection and dose related. The resulting expansion of the marrow lymphocyte population included both immature B cells and null cells, as shown by cell surface and cytoplasmic markers. Similar stimulation of marrow lymphocyte production followed an injection of either bovine serum albumin or mineral oil. No comparable stimulation occurred in either the thymus or the spleen. The results demonstrate that antigens and nonspecific irritants can exert a central effect in the bone marrow, producing a surge in the production of both primary B and non-B lymphocytes. The possible role of external stimulants in determining the normal rate of bone marrow lymphocyte production is discussed.

Regulation of bone marrow lymphocyte production. IV. Cells mediating the stimulation of marrow lymphocyte production by sheep red blood cells: studies in anti-IgM-suppressed mice, athymic mice, and silica-treated mice.

Some cellular requirements have been examined for the stimulation of lymphocyte production in mouse bone marrow by injected sheep red blood cells (SRBC). The increased genesis of marrow lymphocytes after a single dose of SRBC assayed radioautographically after [3H]thymidine labeling was unimpaired in the marrow of mice treated with anti-IgM antibodies from birth to eliminate B lymphocytes, and in congenitally athymic mice lacking T lymphocytes. However, pretreatment of mice with silica to depress macrophage function completely abolished the SRBC effect both on the total lymphocyte production and on the number of B and null small lymphocytes in the marrow. Comparative studies were performed on the thymus and spleen. The results demonstrate that the stimulation of marrow lymphocytes production by SRBC is mediated by a silica-sensitive mechanism, does not require B or T lymphocytes, and is independent of the humoral immune response. Thus, extrinsic agents may amplify the production of primary B cells and other lymphocytes in the bone marrow by an antigen-nonspecific mechanism, putatively mediated by macrophages.

Regulation and localization of lymphocyte production in the bone marrow.

The homeostatic mechanisms which control B lymphocyte renewal in the bone marrow are unknown. Mouse bone marrow produces many small lymphocytes which develop surface IgM and other B lymphocyte properties. Putative precursors show cytoplasmic mu chains but earlier progenitors have been characterized. Some marrow small lymphocytes are long-lived recirculating B and T cells. [3H]Thymidine and IgM labelling in femoral marrow sections suggest that recirculating lymphocytes migrate mainly through the marrow periphery while indigenous lymphocytes may be formed peripherally and migrate centrally as they mature. Thus, the localization of lymphocytes appears to be non-random. The effects of possible regulatory factors on bone marrow small lymphocytes production have been examined by [3H]thymidine labelling and radioautography. Administration of anti-IgM antibodies in vivo eliminates all B lymphocytes but the marrow lymphocyte production rate remains unchanged. After sublethal X-irradiation the marrow shows an over-shoot B lymphocyte production, while the lymphocyte numbers in shielded marrow remain stable. In neonatally thymectomized or congenitally athymic mice marrow lymphocyte production is unaffected. Studies in germ-free and antigen-stimulated mice reveal a basal level of marrow lymphocyte production, normally stimulated non-specifically by environmental factors. Thus, marrow lymphocyte production appears to be basically independent of feedback control from the peripheral B lymphocyte pool or of specific humoral factors, but fluctuates widely after perturbation or when amplified by exogenous stimuli. These findings suggest the importance of microenvironmental factors, as yet undefined, in the regulation of bone marrow lymphocytes.