Hyperdiploid-CD5-positive B cells in mouse.

Hyperdiploid B cells have been found in autoimmune NZB mice as they age. The hyperdiploid cells were found to be clonal both on the basis of cytogenetic analysis and studies of immunoglobulin gene rearrangements at the DNA level. Studies of the inheritance of the hyperdiploid traits in both F1 and backcrosses, as well as NZB recombinant inbred strains, revealed that the presence of hyperdiploid B cells was an inherited recessive trait linked to autoimmune hyperactivity. In addition, hyperdiploid B cells were found to possess a unique chromosome pair which lacked terminal C-bands. This observation allowed analysis of the fate of transferred NZB hyperdiploid B cells into unirradiated recipients. The hyperdiploid B cells were found to expand in recipients and become the dominant population in several lymphoid organs. Spontaneously occurring hyperdiploid B cells were not observed in NZB-xid mice possessing the CBA/N X chromosome, which confers abnormal B cell maturation and results in decreased autoimmunity in NZB-xid mice. Following the discovery that CD5+ B cells were elevated in certain autoimmune states, hyperdiploid B cells were examined and found to be CD5+ B cells as well. The malignant cell in chronic lymphocytic leukemia is also a CD5+ B cell. The hyperdiploid B cells of NZB mice appear to have many of the features of autoimmune B cells, as well as malignant cells.

Influence of host environment on growth of clonal CD5+B (Lyl+B) cells.

Autoimmune NZB mice have increased percentages of CD5+B (Lyl+B) cells in both the spleen and peritoneum. We have previously reported that as NZB mice age they develop a clonal population of hyperdiploid CD5+B cells in the spleen. These cells can readily be transplanted into unirradiated recipients. The growth characteristics of such transplanted hyperdiploid NZB spleen cells were examined in different recipient strains to determine if the immunological status of the host environments affected the growth of the clonal CD5+B cells. Young NZB and NZB.xid recipients (lacking hyperdiploid CD5+B cells) allowed growth and expansion of unpassaged CD5+B cells derived from primary NZB mice. Similarly, (NZBxDBA/2) and (NZBxBALB/c) F1 recipients allowed for expansion of CD5+B cell clones from primary sources. In a separate experiment, T cell-depleted NZB spleen cells containing a hyperdiploid CD5+B cell clone were transferred to SCID mice. The SCID environment supported the growth of the primary clone. None of these recipients normally have elevated CD5+B cells, yet these recipients allowed growth of primary transferred hyperdiploid cells. However, a difference in the ability of these recipient strains in their ability to expand multiply passaged CD5+B cell clones was observed. These results indicate that while hyperdiploid CD5+B cells are difficult to be maintained in culture, they can readily be passaged in vivo. The host environment may provide growth factors or signals for endogenous growth factors. Although the CD5+B clones arise initially in a hyperactive autoimmune environment, a hyperimmune environment is not necessary to support their growth. Transferred CD5+B cells affect the recipient environment and reduce the percentages of normal B cells.

Role of T cells in sex differences in syngeneic bone marrow transfers.

Transferred marrow cells will proliferate in normal mice not exposed to irradiation or any other type of stem cell depletion when five consecutive transfers of 40 million cells are given. Approximately 25% of the mitotic cells are of male donor origin observed cytogenetically in all of the female recipient spleens and marrow analyzed from two weeks to one and one-half years after transfusions. Male donor stem cells are accepted and form a stable component of the self-renewing stem cell pool. In contrast, only 5% female cells are found in male recipients. This sex difference in engraftment is not hormonal since castration of recipients does not alter the percentage of donor cells. Rigorous T depletion of female donor bone marrow, however, increases the percentage of donor engraftment to the level observed when male marrow, either whole or T depleted, is transferred to female recipients. The success of T-depleted female stem cells to seed male recipients is observed in both C57BL/6, a responder strain in which females readily respond to the H-Y antigen as manifest by skin graft rejection, and CBA/J, a strain in which females do not readily respond to H-Y. In addition, recipient nude BALB/c males, which lack a thymus, fail to accept whole bone marrow from BALB/c females. However, male bone marrow cells seed BALB/c nude females. These studies demonstrate that the poor engraftment of female cells in transfused male recipients is abrogated by the removal of T cells from the donor female marrow.

Comparison of response to stem cell differentiation signals between normal and autoimmune mouse strains.

Normal DBA/2 and autoimmune NZB mice were studied with regard to signals eliciting differentiation and division of bone marrow stem cells. Irradiated (NZB X DBA/2)F1 mice were repopulated with various combinations of T-depleted bone marrow from NZB and DBA/2 mice. In response to the repopulation signal of irradiation, recipients of autoimmune NZB marrow initially demonstrated expansion of LY-5+ lymphoid and hemopoietic cells, particularly of the B cell lineage. The greater the proportion of NZB marrow, the higher the percentage of lymphoid cells observed 2 wk post-repopulation. B cells (ThB-positive cells) were increased in disproportionate numbers in recipients of NZB marrow, even those that had received as little as 20% NZB bone marrow cells. However, by 2 mo, the initially observed increase in lymphoid cells in recipients of NZB marrow was no longer observed. Up to 6 mo post-repopulation, cytogenetic analysis revealed that irradiated recipients were repopulated in the same proportion of DBA/2: NZB as was in the injected marrow. Endogenous colony formation assays indicated that recipients of 100% NZB, 80% NZB, and 20% NZB marrow all had greater numbers of splenic endogenous colonies than did recipients of DBA/2 marrow alone. These studies indicated that autoimmune NZB marrow repopulated irradiated mice in the proportion in which it was injected, but there was a disproportionate early increase in cells of the B lineage as well as a disproportionate increase in splenic colony formation.

Enhanced proliferation of transfused marrow and reversal of normal growth inhibition of female marrow in male hosts 2 months after sublethal irradiation.

We have previously shown that bone marrow will seed and proliferate in normal recipients. Transfusion of 50 million cells on each of 4 or 5 consecutive days, a total of 200-250 million cells, resulted in the recipient's marrow being 20-40% of donor origin. The present paper reported on the marked enhancement of proliferation of donor cells in animals that were exposed to sublethal doses of irradiation of 300-900 R. Two months later, when their peripheral blood values had returned to normal, they were transfused with 100 million cells. The number of donor cells in the recipients exposed to 600-900 R reached 55-100% at various intervals after transfusion, with controls averaging 24% and never exceeding 40%. Since the transfused cells numbered less than 40% of the host's own complement of marrow cells, they could not replace 100% of them unless they proliferated more rapidly than the host cells. The implied competitive advantage of the donor cells was ascribed to a reduced capacity for self-renewal of the host's irradiated cells. In recipients exposed to 300 R and in nonirradiated controls, female cells failed to grow in male recipients, while male cells grew as well in female as in male hosts. The inhibition of growth of female cells in the male host was abolished by irradiation with 600 or 900 R, or by the exposure of the female donor cells to anti-Thy-1 serum and complement prior to transfusion. Experiments are under way to test the suggested immunologic nature of the inhibition phenomenon.

Comparison of stem-cell recovery in autoimmune and normal strains.

The capacity of NZB stem cells to proliferate in vivo was evaluated in two systems which required repopulation of peripheral organs. In both types of depletion systems, stem-cell repopulation after cyclophosphamide treatment or adoptive transfer repopulation in lethally irradiated hosts, it was found that NZB stem cells were hyperproliferating. The increase in proliferating cells was most pronounced in the spleens of NZB mice treated with high-dose cyclophosphamide and in lethally irradiated F1 mice reconstituted with NZB T-cell-depleted bone marrow. Thus, upon a stimulus to repopulate, NZB marrow stem cells will hyperproliferate in peripheral organs resulting in an increase in cell number. The abnormality in the marrow cells can be observed in young NZB mice when their marrow cells are in an environment which requires recovery and division.

Cell cycle analysis of lymphocyte activation in normal and autoimmune strains of mice.

The spontaneous spleen cell proliferation and the proliferation induced by in vivo or in vitro stimulation with such polyclonal B cell activators (PBA) as LPS, poly rI.rC, and anti-mu were studied in normal and autoimmune mice. The various murine models of autoimmunity differ in the level of naturally occurring splenic cellular hyperactivity as well as in the ability of their spleen cells to be further stimulated in vitro by polyclonal stimulators. Both the NZB strain and the MRL/Ipr strain had markedly increased numbers and percentages of spontaneously proliferating spleen cells, whereas the BXSB strain did not. Nonautoimmune strains were found to have very small numbers of activated cells in the spleen. However, such normal strains could be induced in vivo to mimic the natural splenic hyperactivity observed in older NZB and MRL/Ipr autoimmune strains by the injection of polyclonal B lymphocyte stimulators. In contrast, old hyperactive NZB mice were not further induced to undergo proliferation by in vivo administration of such stimulators. Density-separated, T depleted, spleen cells of normal and autoimmune mice were stimulated in vitro with PBA in 48-hr cultures. Cells from old MRL/Ipr and NZB mice were abnormal in both the anti-mu response and the LPS response; BXSB mice had normal anti-mu responses. These studies suggest that there is no prerequisite for spontaneous splenic hyperactivity in the development of autoimmunity. In addition, different PBA stimulate separate subsets of B cells that differ in their state of activation in the various autoimmune strains. Finally, different B cell subsets appear to be abnormal in different types of autoimmune mice.

Special proliferative sites are not needed for seeding and proliferation of transfused bone marrow cells in normal syngeneic mice.

The widely held view that transfused bone marrow cells will not proliferate in normal mice, not exposed to irradiation or other forms of bone marrow ablation, was reinvestigated. Forty million bone marrow cells from male donors were given to female recipients on each of 5 consecutive days, 5 to 10 times the number customarily used in the past. When the recipients were examined 2-13 weeks after the last transfusion, donor cells were found to average 16-25% of total marrow cells. Similar percentages of donor cells were found when variants of the enzyme phosphoglycerate kinase determined electrophoretically were used for identification of donor and recipient cells. Evidence is presented that the proportion of donor cells is compatible with a linear dependence on the number of cells transfused over the range tested--i.e., 20-200 million bone marrow cells injected intravenously. Special proliferative sites thus do not appear to be required.

Genetic studies in NZB mice. V. Recombinant inbred lines demonstrate that separate genes control autoimmune phenotype.

The genetic basis for autoimmunity in NZB mice has been investigated through analysis of recombinant inbred lines produced by mating NZB mice with two different non-autoimmune strains. Several genes (at least six) were found to be necessary for the production of eight traits characteristic of the NZB mice that were studied. No fundamental genetic defect (an "autoimmunity gene") was identified that could give rise to the various autoimmune traits studied. This study strongly suggests that NZB disease results from the actions of several separate genes that together result in the characteristic manifestations of autoimmunity.

Analysis of NZB hyperdiploid spleen cells.

In order to study the cell types involved in the hyperdiploidy characteristic of NZB mice, flow cytometric techniques, which measure te DNA content of individual cells, have been used together with standard cytogenetic analysis. The aneuploid cells present in NZB mice were found to be clonally derived. These cells were of large size relative to nonaneuploid cells. They could not be removed by lysis with anti-Thy 1 plus complement nor were they present in the nylon-wool column nonadherent fraction, both of which are characteristic of T cells. By further cytotoxic analysis, the aneuploid cells were found not to express Ly-5 nor NK surface antigens, found on natural killer cells. The aneuploid cells had increased quantities of cell surface H-2 antigen. This marked susceptibility to lysis with anti-H-2 serum was associated with one or more extra copies of chromosome 17, which carries the H-2 complex. A large proportion of these cells also expressed surface Ia and Ig. The aneuploid cells found in the spleens of older NZB mice derive from bone marrow stem cells. Neonatally thymectomized and lethally irradiated NZB X DBA/2 recipients of anti-Thy-1 treated young NZB bone marrow cells ultimately developed aneuploid spleen cells. Such cells are not found in intact or thymectomized NZB Z DBA/2 mice nor in recipients of DBA/2 marrow. Finally, congenic NZB mice carrying the CBZ/N xid failed to develop aneuploidy. Taken together, these studies suggest that the aneuploid cell is a bone marrow-derived stem cell destined to differentiate into the B cell subset lacking in CBA/N mice.

Genetic studies in NZB mice. IV. The effect of sex hormones on the spontaneous production of anti-T cell autoantibodies.

Hybrid NZB X NZW or NZB X DBA/2 females have markedly accelerated development of autoimmunity when compared with their respective male littermates. This difference is attributable to the ability of male sex hormones to retard the expression of autoimmunity. In contrast to the sex differences in expression of autoimmunity in F1 mice, parental NZB males and females have only minor differences in disease expression. We have been investigating the basis for the difference in anti-T cell antibody production between NZB and F1 mice. In this study, the appearance of antibodies cytotoxic for T cells (NTA) was studied in NZB and DBA/2 mice and in their F1 hybrids and backcross progeny. A major sex difference in NTA production was observed in the F1 hybrids; females produced more NTA than did males. Castration of males led to a marked increase in NTA production. Furthermore, the NTA production of castrated male and female F1 mice was significantly suppressed by administration of testosterone in Silastic capsules. In contrast to the studies in F1 mice, we found little difference between intact male and female parental NZB mice at any age studied. Furthermore, NZB mice of both sexes who were given androgen-containing capsules at 2 weeks of age failed to demonstrate a decrease in NTA production. This result suggested an androgen insensitivity in NZB mice with regard to NTA production. This insensitivity, which appears to be a recessive trait, may help to explain why NZB mice do not manifest the sex differences in disease expression observed in the F1 hybrids.

Studies of the effects of sex hormones on autosomal and X-linked genetic control of induced and spontaneous antibody production.

In these studies we investigated the modifying effect of sex hormones on both the levels of induced antibodies after immunization with single-stranded DNA (ssDNA) and the levels of spontaneously produced anti-T cell antibodies (NTA). To learn whether the responses were genetically determined or under hormonal regulation, we analyzed hybrids produced by crossing the autoimmune NZB strain with the nonautoimmune DBA/2 strain. For both anti-ssDNA and NTA, males usually had a lower response than females; this difference could largely be removed by castration of the males. Females given testosterone implants also had decreased antibody levels. The higher responses in females and suppression by testosterone were true for all mice studied except NZB mice. NZB mice appear to have an insensitivity to the suppressive effects of testosterone.

Transplantation of murine bone marrow without prior host irradiation.

The experiments presented test the hypothesis that pluripotential stem cells (assayed in the mouse as CFU-S) are normally not in cycle and that the failure of normal marrow transfusions to take in normal recipients is due to the absence of a stimulus to turn CFU-S into cycle. Following marrow transfusion from male donors into female isogeneic recipients, spleen, liver, and various parts of the skeleton were shielded to protect transfused donor cells from lethal doses of radiation gives to the rest of the body. Percentages of hemopoietic donor and host cells were subsequently determined by karyotyping C banded marrow and spleen metaphases and identifying of Y chromosome. The results support the notion that the failure of normal marrow to take in normal recipients is not due to inadequate numbers of transfused cells. Permanent colonization by donor cells, however, requires not only triggering CFU-S into cycle, but also emptying of 'niches' normally occupied by endogenous CFU-S. Partial body radiation meets both requirements. In addition, the results indicate that recently arrived donor cells, protected in the shielded portion of the body, seed more readily into the irradiated areas of the skeleton than do similarly protected host cells.

Cytogenetic studies of a male with sporadic intestinal lymphangiectasia: 45,X/46,XY mosaicism with pseudo- and hyperdiploid subpopulations in cultured tissues.

45,X/46,XY mosaicism was found in peripheral blood, bone marrow, and tissue cultures of an adult male with intestinal lymphangiectasia (IL). Turner phenotype was not present; his meiotic metaphase analysis was normak, and his dermatoglyphics resembled those of his family. Ten separate tissue culture lines from three biopsies of skin and thyroid gland contained 45,X cells (14.8 to 78.3%). Autosomal aneuploidy, resulting in pseudo- or hyperdiploidy, was also present in 4.3 to 41.6% of the cells. A hyperdiploid clone with a 47,X,+10,+18 karyotype was found in 22.6% of cells in one line. A second hyperdiploid clone with a 48,X,+2,+18,+18 karyotype occurred in 7.6% of cells from another line containing a total of 41.6% pseudo- and hyperdiploid cells. Such clonal abnormalities were not typical of tissue cultures from other patients done in our laboratory. Growth of our patient's tissue cultures was subnormal, and none proliferated beyond the fourth subculture. The significance of this observation remains to be determined. Our results do not allow us to conclude whether our patient's mosaicism of somatic tissues arose during embryogenesis, or whether it originated post-natally. The secondary immunodeficiency which occurs in IL may explain persistence of cells with unusual combinations of autosomal aneuploidy in our patient's tissues.

Genetic studies in NZB mice. II. Hyperdiploidy in the spleen of NZB mice and their hybrids.

The presence of hyperdiploidy was studied in New Zealand black (NZB) mice and the progeny of NZB X DBA/2 crosses and backcrosses. Hyperdiploidy was observed in the spleens of a majority of NZB mice but not in DBA/2 mice at 1 year of age. In crosses of NZB with the DBA/2 strain, hyperploidy was observed only in backcrosses to NZB. Hyperdiploidy appeared to be determined by a recessivley inherited trait and was not related to the presence of other immunological abnormalities, including splenomegaly, hypergammaglobulinemia, and spontaneous antibodies cytotoxic for T cells and reactive with single-stranded DNA. Abnormal cells were not present in Concanavalin A-stimulated 48-h spleen cultures. There was no difference in the in vitro sister chromatid exchange rate between the autoimmune NZB strain and the non-autoimmune DBA/2 strain. Identification of NZB chromosomes by banding analysis showed that chromosomes 15 and 17 were frequently present in more than two copies in hyperdiploid spleen cells. NZB chromsomes also had reduced C-banding in an autosomal pair. These studies indicate that chromosomal abnormalities which occur in NZB mice may be useful as genetic and cytogenetic markers.

Genetic studies in NZB mice. I. Spontaneous autoantibody production.

The appearance of naturally occurring thymocytotoxic autoantibodies (NTA) and spontaneously produced antibodies to single-stranded DNA (ssDNA) was studied in NZB, and DBA/2 mice and their F1 and backcross progeny. NTA production was markedly decreased in males; however, castrated males produced quantities of NTA similar to those of females. Because the amount of NTA was influenced by sex hormones, it was necessary to gonadectomize all progeny to determine the mode of inheritance. Such studies suggested that NTA production was determined by a single locus with a gene dosage (codominant) mode of expression. The spontaneous production of antibodies to ssDNA appeared to be inherited as a single dominant genetic trait. The quantity of anti-ssDNA was also found to be under additional regulation; either a gene dosage effect or more likely a regulatory gene. The genes controlling the presence and quantity of ssDNA antibodies were not linked to the gene controlling the appearance of NTA.