Unexpected rearrangement and expression of the immunoglobulin lambda1 locus in scid mice.

In severe combined immunodeficient (scid) mice, V(D)J recombination is severely impaired due to a recessive mutation (scid). Thus, we were surprised to find in this study that Vlambda1-Jlambda1 rearrangement is routinely detectable in scid fetal liver, adult bone marrow, and spleen in the apparent absence of completed VH-DJH and Vkappa-Jkappa rearrangements. Particularly surprising, we found the level of Vlambda1-Jlambda1 rearrangement in scid fetal liver to be comparable to that in fetal liver of wild-type mice. The majority of scid Vlambda1-Jlambda1 rearrangements contained abnormal deletions at the VJ junction, consistent with the known effect of scid. However, approximately 15% of Vlambda1-Jlambda1 rearrangements lacked abnormal deletions. Productive lambda1 transcripts resulting from in-frame rearrangements were readily detectable in scid adult bone marrow and spleen, consistent with our ability to detect lambda1-expressing cells by flow cytometry in the spleens of bcl-2-transgenic scid mice. Strikingly, lambda1 transcripts from individual scid mice often showed VJ junctional sequences with the same recurring palindromic (P) additions of three, four, or five nucleotides. To account for these findings, we suggest that (a) nonhomologous end joining of Vlambda1 and Jlambda1 coding ends in fetal B lineage cells may not be (severely) impaired by scid; (b) recurring P additions in scid lambda1 transcripts may reflect certain molecular constraints imposed by scid on the resolution of Vlambda1 and Jlambda1 hairpin coding ends; and (c), scid lymphocytes with productively rearranged Vlambda1 and Jlambda1 elements may differentiate into recombinase-inactive cells and emigrate from bone marrow to spleen.

Differential effect of an Ig mu transgene on development of pre-B cells in fetal and adult SCID mice.

Progression of pro-B lymphocytes to the pre-B stage depends on the expression of a pre-B cell receptor (pre-BCR), consisting of an Ig mu H chain, Ig surrogate light chain, and associated signal transducing chains. Mice that are unable to express a pre-BCR show an arrest of B cell development at the pro-B stage. Such is the case for severe combined immune deficient (SCID) mice in which mu chains are not made because of a defect in V(D)J recombination. When mu chains are made, as in SCID mice bearing a functional mu transgene, then B cell differentiation can proceed to the pre-B stage. However, as reported here, a mu transgene (M54) that promotes development of SCID pre-B cells in adult bone marrow fails to do so in fetal liver. We suggest that a pre-BCR containing the M54 mu chain cannot signal progression of pro-B cells to the pre-B stage in the fetal liver microenvironment.

Extended duration of DH-JH rearrangement in immunoglobulin heavy chain transgenic mice: implications for regulation of allelic exclusion.

Here we show that suppression of VH-DJH rearrangement in mice bearing a mu heavy (H) chain transgene (mu-tg mice) is associated with an extended period of DH-JH rearrangement, the first step of Immunoglobulin H chain gene rearrangement. Whereas DH-JH rearrangement is normally initiated and completed at the pro-B cell stage, in mu-tg mice it continues beyond this stage and occurs most frequently at the small (late) pre-B stage. Despite ongoing DH-JH rearrangement in late pre-B cells of mu-tg mice, VH-DJH rearrangement is not detectable in these cells. We infer that the lack of VH-DJH rearrangement primarily reflects tg-induced acceleration of B cell differentiation past the stage at which rearrangement of VH elements is permissible. In support of this inference, we find that the normal representation of early B lineage subsets is markedly altered in mu-tg mice. We suggest that the effect of a productive VH-DJH rearrangement at an endogenous H chain allele may be similar to that of a mu-tg; i.e., cells that make a productive VH-DJH rearrangement on the first attempt rapidly progress to a developmental stage that precludes VH-DJH rearrangement at the other allele (allelic exclusion).

Subunit composition of pre-T cell receptor complexes expressed by primary thymocytes: CD3 delta is physically associated but not functionally required.

Maturation of immature CD4-CD8- (DN) thymocytes to the CD4+CD8+ (DP) stage of development is driven by signals transduced through a pre-T cell receptor (TCR) complex, whose hallmark is a novel subunit termed pre-T alpha (pT alpha). However, the precise role of pre-TCRs in mediating the DN to DP transition remains unclear. Moreover, progress in understanding pre-TCR function has been hampered thus far because previous attempts to demonstrate expression of pT alpha-containing pre-TCRs on the surface of normal thymocytes have been unsuccessful. In this report, we demonstrate for the first time that pT alpha-containing pre-TCR complexes are expressed at low levels on the surface of primary thymocytes and that these pre-TCR complexes comprise a disulfide-linked pT alpha-TCR-beta heterodimer associated not only with CD3-gamma and -epsilon, as previously reported, but also with zeta and delta. Interestingly, while CD3-delta is associated with the pre-TCR complex, it is not required for pre-TCR function, as evidenced by the generation of normal numbers of DP thymocytes in CD3-delta-deficient mice. The fact that any of the signaling components of the pre-TCR are dispensable for pre-TCR function is indeed surprising, given that few pre-TCR complexes are actually expressed on the surface of primary thymocytes in vivo. Thus, pre-TCRs do not require the full array of TCR-associated signaling subunits (gamma, delta, epsilon, and zeta), possibly because pT alpha itself possesses signaling capabilities.

Development of B cells in scid mice with immunoglobulin transgenes: implications for the control of V(D)J recombination.

The inability of scid pro-B cells to progress to the pre-B and B cell stages is believed to be caused by a defective recombinase activity that fails to resolve chromosomal breaks resulting from attempted V(D)J recombination. In support of this model, we report that certain immunoglobulin transgenes, specifically those which strongly inhibit endogenous VH-to-DJH and V kappa-to-J kappa rearrangement in wild-type mice, allow scid pro-B cells to progress to the pre-B and B cell stages. This rescue of scid B cell differentiation is associated with a dramatic reduction in expression of the recombination activation genes, RAG1 and RAG2, and with reduced transcription of the kappa locus.

The defect in murine severe combined immune deficiency: joining of signal sequences but not coding segments in V(D)J recombination.

Pre-B and pre-T cell lines from mutant mice with severe combined immune deficiency (scid mice) were transfected with plasmids that contained recombination signal sequences of antigen receptor gene elements (V, D, and J). Recovered plasmids were tested for possible recombination of signal sequences and/or the adjacent (coding) sequences. Signal ends were joined, but recombination was abnormal in that half of the recombinants had lost nucleotides from one or both signals. Coding ends were not joined at all in either deletional or inversional V(D)J recombination reactions. However, coding ends were able to participate in alternative reactions. The failure of coding joint formation in scid pre-B and pre-T cells appears sufficient to explain the absence of immunoglobulin or T cell receptor production in scid mice.

Transcription of unrearranged antigen receptor genes in scid mice.

The scid mouse mutant is severely deficient in lymphocytes; cells of the B or T lymphocyte lineage cannot be detected by either serological or functional assays. However, as shown here, germ-line transcripts of B cell immunoglobulin (Ig) constant and variable region genes and of T cell receptor (TCR) genes are detectable in lymphopoietic tissues of scid mice, as well as B and T lineage-specific lambda 5 and T3 delta transcripts. We conclude that B and T lineage-committed cells do arise in scid mice and that their Ig and TCR genes are accessible to enzymes involved in their recombination. This suggests that scid impairs lymphopoiesis at the stage at which antigen receptor genes normally undergo rearrangement.

Pathology of Listeria infection in murine severe combined immunodeficiency. A study by immunohistochemistry and electron microscopy.

Mice bearing the scid mutation lack functional T and B lymphocytes and partially resist infection with the intracellular pathogen Listeria monocytogenes. In this study, we describe the histopathologic features of the infection in scid mice. Infected scid showed hyperplasia of mononuclear cells in lymphoid organs without any evidence of granulomas nor of lymphoid cell development. Mononuclear phagocytes in various tissues of infected scid mice had markedly enhanced expression of class II histocompatibility molecules (or Ia molecules). Furthermore, Ia expression was also found in endothelial and epithelial cells of many organs in the later stages of infection. These results confirm that scid mice have a mechanism for activation of Ia antigen expression, which is independent of lymphocyte function.

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.

Evidence of functional lymphocytes in some (leaky) scid mice.

Although the majority of severe combined immune deficiency (scid) mice lack functional lymphocytes, some (2-23%) appear to develop a limited number of B and T cells between 3 and 9 mo old. Most of these leaky scid mice were shown to contain very few clones (less than or equal to 3) of Ig-producing plasmacytes. Clonal progeny were distributed unevenly in the lymphatic tissues and appeared as discrete plasmacytic foci. In many cases, individual clones persisted for several months and produced abnormally high concentrations of Ig that included multiple isotypes. Functional T cells were inferred from the ability of leaky mice to reject allogeneic skin grafts, a T cell-dependent reaction. Interestingly, approximately 40% of leaky mice developed thymic lymphomas. In other respects, leaky mice resembled regular scid mice; e.g., their splenic cells failed to express common lymphocyte antigens (Ly-5[B220], Ly-1) and to proliferate in response to lymphocyte mitogens. Histologically, their lymphoid tissues retained the same general pattern of severe lymphocytic deficiency as scid mice.

A T cell-independent mechanism of macrophage activation by interferon-gamma.

A primary interest in immunity to intracellular pathogenic microorganisms and tumors is to understand the mechanisms by which macrophages are activated for various functions. Two parameters of macrophage activation are the expression of the class II histocompatibility proteins or Ia molecules (1), and cytotoxic activity. The ability of T cells to induce these responses has been extensively documented and occurs via their secretion of interferon-gamma (IFN-gamma) after interaction with antigen (2-6). However, in a recent study using mice with the severe combined immunodeficiency (scid) mutation (7) which have no detectable T or B cell functions (7-9), we were surprised to find the induction of Ia expression on macrophages and the partial inhibition of bacterial growth after infection with Listeria monocytogenes (10). We have now utilized neutralizing monoclonal antibodies specific for murine IFN-gamma to investigate the mechanism of macrophage activation in scid mice. We show here that IFN-gamma can be produced by scid mice in the absence of lymphocyte-mediated immunity, and this IFN-gamma is important for macrophage activation during infection with Listeria. These results indicate the presence of an important T lymphocyte-independent mechanism of macrophage activation and IFN-gamma production in response to infection.

Regulation of macrophage Ia expression in mice with severe combined immunodeficiency: induction of Ia expression by a T cell-independent mechanism.

We have studied the expression of Ia molecules by macrophages from mice with severe combined immunodeficiency (CB-17 scid) that lack demonstrable T cell and B cell functions. CB-17 scid mice had approximately normal numbers of Ia-bearing macrophages in the peritoneal cavity, spleen, and liver. Peritoneal macrophages responded in culture to T cell-derived lymphokines with enhanced expression of Ia molecules. However, unlike immunocompetent controls, SCID mice could not enhance Ia expression in an antigen-specific T cell-dependent manner after secondary challenge in vivo with a conventional protein antigen such as hemocyanin. Further demonstration of their T cell deficiency was the failure of CB-17 scid spleen cells to proliferate and produce IL 2 in response to the T cell mitogen, concanavalin A. Upon infection with Listeria monocytogenes, CB-17 scid mice developed chronically high loads of bacteria, whereas CB-17 control mice eliminated all viable bacteria and became resistant to secondary infection. However, Listeria-infected CB-17 scid mice did show, in parallel with the CB-17 controls, an unexpected and striking increase of Ia-positive macrophages. These data indicate that induction of Ia expression in macrophages can occur via a mechanism that is independent of mature T cells.

Transplantable progenitors of natural killer cells are distinct from those of T and B lymphocytes.

We have utilized a mouse mutant (C.B-17 scid) that lacks functional T and B lymphocytes to examine the relationship among transplantable progenitors of natural killer (NK) cells, T cells, and B cells. The NK-progenitor cells contained in the bone marrow were detected by their ability to generate mature NK cells, following transfer of bone marrow cells into NK cell-depleted and lethally irradiated mice. Regeneration of NK activity in the recipient mice was monitored by two different assays: the ability to rapidly clear infused YAC-1 cells in vivo and the ability of spleen cells to lyse YAC-1 cells in vitro. Recipients were also tested for the presence of mitogen-responsive T and B cells and for prethymocytes (thymus-repopulating cells). We found that the capacity of C.B-17 scid bone marrow cells to generate mature NK cells was equivalent to that of control C.B-17 bone marrow cells. The regenerated NK cells shared similar functional activity and surface phenotype. In contrast, bone marrow cells from C.B-17 scid mice failed to generate thymocytes and peripheral T and B cells. These data indicate that the transplantable NK-progenitor cells are not defective or deficient in C.B-17 scid mice and, therefore, are distinct from the transplantable progenitor(s) of T and B cells.

Severe combined immunodeficiency (SCID) in the mouse. Pathology, reconstitution, neoplasms.

Histologic findings in mice with severe combined immunodeficiency (SCID) were remarkably uniform, consisting of lymphopenia, a rudimentary thymic medulla without cortex, relatively empty splenic follicles and lymph nodes, and undeveloped bronchial and gastrointestinal lymphocytic foci. Fluorescence-activated cell sorter studies revealed a few T cells (apparently nonfunctional) in thymus and spleen; interestingly, these cells seemed highly disposed to neoplasia, because thymic T-cell lymphomas were observed in 41 of 269 mice. No pre-B or B cells could be identified. Cells of the myeloid lineage appeared normal. Reconstitution of lymphoid tissues was achieved after intravenous injection of histocompatible bone marrow cells.

Expression of murine Lm-1 locus. Lm-1 determinants on lymphocytes and macrophages, and effects of Lm-1 incompatibility on bone marrow grafts.

Lm-1 is an Igh-linked locus that codes for cell surface alloantigens (Lm-1 determinants) recognized by T lymphocytes. Using Lm-1 congenic strains and cold-target inhibition of anti-Lm-1-specific lysis by cytotoxic T lymphocytes, we were able to demonstrate differential expression of two distinct Lm-1 antigenic determinants. One determinant is expressed on the surface of T cell blasts, the other on a number of pre-B cell lines. Both determinants are present on B cell blasts. Macrophages also bear Lm-1 determinants, and possibly express a determinant not found on lymphocytes. Fibroblasts, (unstimulated) thymocytes, and immature T cells lack detectable Lm-1 determinants. These data indicate that expression of the Lm-1 locus is dependent on cell lineage and the stage of cell differentiation or activation. We propose that Lm-1 is a lymphocyte-macrophage differentiation locus containing a number of structurally and functionally related genes. Evidence was presented that Lm-1 may also serve as a histocompatibility locus of major importance for bone marrow transplantation. Specifically, when Lm-1-incompatible bone marrow cells and spleen cells (from normal or anti-Lm-1 immune mice) were transplanted into X-irradiated recipients, the maturation and/or function of bone marrow-derived donor B cells was delayed or inhibited.

Functional status of cells from lymphoid and myeloid tissues in mice with severe combined immunodeficiency disease.

Cells from mice with severe combined immunodeficiency disease (SCID) were tested in assays that measure myeloid and lymphoid function. Results showed that C.B-17 scid and their normal counterparts (C.B-17) have similar levels of spleen colony-forming units. The frequency of in vitro myeloid colony-forming units in C.B-17 scid spleen is elevated, but the absolute number of colony-forming units in C.B-17 scid and C.B-17 spleen is similar. The absolute number of bone marrow colony-forming units in C.B-17 scid and C.B-17 mice is comparable. Cells from C.B-17 scid spleen are consistently negative in all tests of B and T cell function. C.B-17 scid splenocytes fail to proliferate in response to T and B cell mitogens or to allogeneic lymphocytes in a one-way MLR; C.B-17 scid cells do serve as stimulators in MLR. B lymphocyte colony-forming units are absent, as are cytotoxic lymphocyte precursors and cells that can generate T cell colonies with cytotoxic progenitors. The microenvironment of the C.B-17 scid mouse is conducive to lymphocyte differentiation, because functional B and T cells are easily detectable in mice reconstituted with normal bone marrow cells. The results of this study indicate that scid specifically impairs the differentiation of stem cells into mature lymphocytes; myeloid cell differentiation is not affected.