Homogeneous immunoglobulins in the serum of irradiated and bone marrow reconstituted mice: the role of thymus and spleen.

The influence of thymectomy and splenectomy on the frequency and class distribution of homogeneous immunoglobulins (H-Ig) in serum was studied in lethally irradiated (DBA/2 x C57Bl/Rij)F1 mice reconstituted with syngeneic bone marrow. During four follow-up periods in the first 9 months after transplantation, the sham-operated controls and splenectomized animals developed transient H-Ig in an average frequency of 14.2 and 15.7% respectively. There were no marked differences in the incidence of H-Ig within these two groups. In contrast, thymectomized mice and mice both thymectomized and splenectomized showed H-Ig in much higher frequencies (average percentages 31.6 and 36.5, respectively). The highest frequency of H-Ig was observed between 1.5 and 3.5 months after transplantation. H-Ig of the IgG1 and IgG2 subclasses were most frequent in all groups during the first 3.5 months. Later, H-Ig belonging to the IgM class also appeared in somewhat higher numbers. H-Ig of the IgA class was a very rare finding at any time. These results indicate that the presence of the thymus, but not necessarily of the spleen, is an important factor in the regulation of the immunoglobulin heterogeneity during the reconstitution of the immune system in lethally irradiated and bone marrow reconstituted mice.     

Effect of Biostim (RU 41.740) on natural killer cell generation from bone marrow precursors

We have evaluated the possible effect of RU 41.740 (Biostim), a mixture of two glycoproteins extracted from K. pneumoniae, on the in vitro interleukin-2 (IL-2)-induced generation of NK cells from bone marrow (BM) precursors and on the in vivo reconstitution of splenic NK activity in lethally irradiated (9 Gy) and BM reconstituted mice. Our results show that RU 41.740 is able to augment the generation of NK cells when added (1-0.01 micrograms/ml) to normal or 5-fluorouracil-resistant BM, cultured in the presence of recombinant IL-2. Also, in vivo treatment of lethally irradiated mice, transplanted with syngeneic BM cells, with RU 41.740 (1-0.1 mg/kg i.v.) from day 0 through day 4 after BM graft, resulted in a significant augmentation of NK activity reconstitution.     

Splenectomy influences homing of transplanted stem cells in bone marrow-ablated mice

Cell mobilization, a process that influences circulation, margination, and finally, homing play key roles in the regeneration processes mediated by stem cells. Recent studies as well as prior studies from our group indicate an important role of the spleen in hematopoietic reconstitution, but to date the role of the spleen in hematopoietic reconstitution has been unclear and it has not been precisely documented in ablated animals. Therefore, we undertook the present study to define more closely the role of the spleen in hematopoietic reconstitution in lethally irradiated mice. After transplantation of irradiated mice with lacZ+ -marked lin- / CD117+ bone marrow cells, we compared splenectomized mice (T(S), splenectomy performed prior to irradiation) to nonsplenectomized, irradiated mice (T(N)) as well as to normal (unirradiated) mice. Impaired hematopoietic reconstitution was observed in T(S) mice. Splenectomy markedly altered the distribution of hematopoietic stem cells, as demonstrated by fluorescence-activated cell sorting analysis of endogenous CD117+ cells in the thymus and bone marrow of recipients. Cell engraftment was demonstrated by histochemical and polymerase chain reaction analyses of recipient tissues. These experiments demonstrated that in T(S) animals, transplanted hematopoietic stem cells mobilized to extravascular tissues, particularly the gastrointestinal tract. The number of donor cells in recipient tissues continued to increase for 30 days after transplantation with the highest numbers observed in the T(S) group. DNA marking analysis led to the conclusion that engrafted cells were not only integrated into recipient tissues but were also capable of performing complex cellular processes, including proliferation and repair. Our results are consistent with the novel possibility that cellular repair markedly affects stem cell regenerative functions and that repair is markedly influenced by the integrity and presence of organs not directly involved in specific tissue regeneration processes, particularly the spleen.     

Regulatory T cells control autoimmunity following syngeneic bone marrow transplantation

Sublethally irradiated, immunodeficient, C57BL/6 RAG-2 gene-deleted recipient mice reconstituted with T cell-depleted bone marrow (BM) grafts frequently developed diarrhea, lost weight and showed signs of autoimmunity, dying between 4 and 7 weeks after reconstitution. Mice died despite evidence of efficient donor-derived hemato-lymphoid reconstitution, and disease was associated with the presence of IgG anti-nuclear antibodies. Autoimmunity was initiated by T cells, but could be prevented by transfer of naturally arising regulatory T cells. In contrast, lethally irradiated, BM-reconstituted immunocompetent, C57BL/6 mice survived without signs of autoimmunity. Survival of immunocompetent mice was shown to be due to the presence of residual, extra-thymically located, radio-resistant, functional regulatory T cells. The importance of regulatory T cells was further shown by the reduced survival of immunocompetent BM recipients whose CD25+ T cells had been depleted prior to bone marrow transplantation. The implications of these results in the context of syngeneic graft-versus-host disease following BM transplantation are discussed.     

Syngeneic preference manifested by thymic stroma during development of thymocytes from bone marrow cells

The question whether major histocompatibility complex (MHC) recognition is expressed in interactions between thymocyte progenitors and thymic stroma cells was investigated in an organ culture system, in which inductive interactions between thymic stroma cells and thymocyte progenitors of different MHC haplotypes could be measured. Thymocyte-depleted fetal thymuses were reconstituted with mixtures of syngeneic and allogeneic bone marrow cells, which also differed in their Thy-1 allele. The relative repopulating ability of the cells was estimated by determining the percentage of emerging Thy-1.1+ vs. Thy-1.2+ thymocytes. Similar values of Thy-1+ cells of the bone marrow donor type developed when the thymus were reconstituted by bone marrow from donors which were either syngeneic or allogeneic to the thymic explants. However, when a 1:1 mixture of syngeneic and allogeneic cells was applied to the thymus, a syngeneic preference was manifested in development of Thy-1+ cells. When mixtures of bone marrow cells from C57BL/Ka (Thy-1.1) and B10.A MHC-congenic (Thy-1.2) mice were used, this developmental preference was found to map to the I-E region. Thymocytes derived from bone marrow cells allogeneic to the stroma, seeded on their own, manifested an advantage over allogeneic bone marrow cells from a different MHC haplotype, in a secondary reconstitution. This suggested that allogeneic bone marrow progenitor cells can be "educated" by the host thymic stroma to behave, in the competitive reconstitution, like syngeneic cells.     

Induction of immunological tolerance by chimeric thymus transplantation

The role of bone marrow derived cells (BMD) within the thymus during the induction of immunological tolerance was investigated using transplantation of chimeric thymuses. Chimeric thymuses were constructed by reconstituting lethally irradiated BALB/C recipients with T-cell depleted C3H bone marrow (BM) cells. Two to three months post bone marrow transplantation the thymuses of these animals, in which the epithelium was of BALB/C origin and BMD cells of C3H origin (verified by immunoperoxidase staining), were transplanted into thymectomized, lethally irradiated BALB/C recipients which were reconstituted with T-cell depleted syngeneic (BALB/C) bone marrow cells. Induction of specific tolerance to the BMD cells (C3H origin) present in the chimeric thymus could be demonstrated in MLR at 3 to 6 months post chimeric thymus transplantation. It is concluded that bone marrow derived cells are able to induce immunological tolerance within the thymus.     

Green fluorescent protein-transgenic mice: immune functions and their application to studies of lymphocyte development

Green fluorescent protein (GFP) transgenic (GFP+) mice express GFP in most tissues except erythrocytes and hair. Immune responses of GFP+ mouse and their application to studies of lymphocyte development were investigated. Flow cytometric analyses revealed that differentiation patterns of lymphocytes from GFP+ mice are equivalent to those from parental C57BL/6 mice. There was no difference in mature T-cell proliferative ability in response to allogeneic stimulator cells or anti-CD3epsilon stimulation between GFP+ and C57BL/6 mice. Furthermore, the anti-OVA antibody response of GFP+ mice was also the same as that of C57BL/6 mice. Taken together, these results show no immunological differences between GFP+ and C57BL/6 mice. Bone marrow transplantation and in vitro thymus reconstitution experiments were performed in an attempt to apply the GFP+ mice to the analysis of lymphocyte development. When bone marrow cells from GFP+ mice were transplanted. T and B lymphocytes containing GFP developed normally in scid recipients. Next we examined intrathymic T-cell development by hanging drop culture methods. GFP+ and CD4+8+ immature T-cells developed normally from bone marrow cells in the reconstituted thymus. The experimental system using hematopoietic cells from GFP+ mice is a powerful tool for visualizing lymphocyte development.     

Resistance to lymphoid engraftment in lpr recipients of normal bone marrow: characterization of chimeric stem cell, monocyte and peripheral lymphoid lineages

Lethally irradiated MRL/lpr mice reconstituted with T cell-depleted bone marrow stem cells from the non-autoimmune strain A. Thy had been shown to develop a state of long-term split chimerism; erythrocytes were derived from the A. Thy donor, while peripheral lymphocytes were derived from the lpr recipient. In contrast, recipients of the non-lpr-congenic strain, MRL/+, were fully repopulated in both lineages by donor-derived hematopoietic cells. In order to more fully understand the mechanisms responsible for this type of split chimerism, we have investigated additional genetic and developmental parameters. We found that histocompatible normal B cell precursors engrafted C3H/HeJ and C57BL/6/+ mice much better than they engrafted the corresponding lpr congenic strains, indicating that resistance to lymphoid engraftment was not unique to the MRL background. Bone marrow cells and peritoneal macrophages were found to express the donor H-2 phenotype in both non-lpr and lpr recipients, limiting resistance to the lymphoid lineage. Moreover, we showed that normal bone marrow stem cells passaged in an lpr host environment were subsequently able to repopulate the B cell lineage of non-lpr secondary recipients, proving that prelymphoid stem cells were intact. Although lymph node cells from A. Thy----MRL/lpr chimeras were lpr-derived, they did not show the abnormal surface marker expression associated with the lpr phenotype, nor did they develop lymphoid hyperplasia or elevated autoantibody levels. However, A. Thy----MRL/lpr chimeras differed from normal mice in that their spleens were markedly deficient in IgM+ B cells.     

Immunoglobulin-bearing B cells reconstitute and maintain the murine Ly-1 B cell lineage

Previous reconstitution studies in irradiated mice distinguished Ly-1 B from other B cells. The predominant B cell populations in spleen and lymph node are readily reconstituted from progenitors present in adult bone marrow. Ly-1 B cells, in contrast, are reconstituted from cells in peritoneum rather than bone marrow. Further studies, presented here, demonstrate that Ly-1 B populations are reconstituted from peritoneal cells that already express both surface immunoglobulin (IgM) and Ly-1, that is, Ly-1 B. Such cells reconstitute and maintain the Ly-1 B population in irradiated recipients for at least 6 months.     

The development of NK cell activity in thymectomized bone marrow chimaeras.

Most of the natural killer (NK) cells, recently derived from the bone marrow (14 days after injection of bone marrow cells into lethally irradiated mice), express the Thy-1 antigen; after some period of time (by day 28) a normal proportion (50%) of Thy-1+ NK cells is found. This study demonstrates that the shift of these Thy-1+ NK cells to Thy-1- NK cells is influenced by the thymus: NK cells developing in the total absence of the thymus--bone marrow (bm) cells from adult thymectomized (Tx) or nude (nu) mice injected into thymectomized recipients, (Tx) bm----(Tx) or nu bm----(Tx)--remain Thy-1+, while the presence of the thymus at some stage of the NK cell maturation--bone marrow cells injected into thymectomized recipients, bm----(Tx), or normal recipients reconstituted with bone marrow cells from thymectomized or nude mice, (Tx) bm----or nu bm----normal recipients--is sufficient for the development of normal ratio of Thy-1+ and Thy-1- NK cells.     

Double mutant MRL-lpr/lpr-gld/gld cells fail to trigger lpr-graft-versus-host disease in syngeneic wild-type recipient mice, but can induce wild-type B cells to make autoantibody

Lethally irradiated mice reconstituted with histocompatible stem cells from Fas-deficient MRL/lpr mice develop a wasting syndrome reminiscent of chronic graft-versus-host disease. However, reconstitution with double Fas-/Fas ligand (FasL)-deficient stem cells does not result in wasting disease, demonstrating that FasL expression is an important component of the effector mechanisms leading to this syndrome. In the absence of wasting disease double-deficient T cells can induce wild-type B cells to make autoantibodies. These data indicate that autoantibody production is regulated by FasL-expressing T cells, and that Fas-sufficient wild-type B cells differ from Fas-deficient Ipr cells only with regard to their sensitivity to FasL.     

Partial reconstitution of V(D)J rearrangement and lymphocyte development in RAG-deficient mice expressing inducible, tetracycline-regulated RAG transgenes

Previously, we described a tetracycline-based autoregulatory system for inducible gene expression in mammalian cells and transgenic mice [Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 6522]. We have tested the ability of this system to drive functional expression in vivo of the V(D)J recombination activating genes, RAG1 and RAG2. In induced transgenic mice, transgenic RAG1 and RAG2 mRNA is observed in thymus and spleen, and expression of both transgenes on the RAG1 or RAG2 knockout backgrounds allows partial, inducible, lymphocyte reconstitution. In thymus and peripheral lymphoid organs of reconstituted animals, cells expressing CD4 and/or CD8 on their surface, also express CD3 and TCR beta chain. In these animals, V(D)J rearrangements are detected in thymus, lymph nodes, and spleen at the TRB locus, and in thymus and lymph nodes at the TRD locus. At the TRA locus, broken ends at V(D)J recombination signals are detected only in thymus, as are reciprocal signal joint products derived from deletional rearrangement. T cell reconstitution occurs in these animals whether they are induced in utero during development, or shortly after birth. A low level of B cell reconstitution is also observed. B220+IgM+ cells are observed in spleen only in induced animals, and rearrangements at IGH and IGK loci are detected in bone marrow and spleen. Broken signal ends at the IGK locus, are not detected in peripheral lymphoid organs. Inducible reconstitution of normal levels of serum immunoglobulin, including heavy chain class switch isotype variants is also observed in these animals. Further, these transgenes do not appear to interfere with lymphocyte development mediated by functionally rearranged TRB chain or IGH chain transgenes in RAG-deficient animals. These mice provide a unique system for the inducible activation of V(D)J recombination and the development of primary lymphocytes.     

High-resolution imaging and antitumor effects of GFP(+) bone marrow-derived cells homing to syngeneic mouse colon tumors

Bone marrow-derived cells (BMDCs) participate in the growth and spread of tumors of the breast, brain, lung, and stomach. To date, there are limited reports of bone marrow involvement in colon cancer pathogenesis, but such findings would have the potential to generate novel treatments for colon cancer patients. We have established a mouse model for imaging BMDCs from whole tumor to single-cell resolution, whereby the bone marrow of lethally irradiated host animals is reconstituted with EGFP-expressing bone marrow cells from matched TgActb(EGFP) donors. The BM transplants yield mice with fluorescently labeled bone marrow, and so BMDCs can subsequently be monitored within a tumor through optical imaging. Successful BM reconstitution was confirmed at 8 weeks after transplantation, when surviving BALB/c mice were injected with CT26 mouse colon cancer cells. We find that up to 45% of cells dissociated from the tumors are GFP(+) and approximately 50% of Lin(+), CD11b(+), and CD3(+) cells express high levels of GFP. Notably, tumor growth is reduced in BM transplanted animals, compared with untransplanted host mice or EGFP-expressing BM donor mice. A needed next step is to separate the molecular and cellular (eg, T cells, NK cells, macrophages) bases of the antitumor effect of the BMDCs from any protumorigenic effect that could be subverted for therapeutic gain.     

Functional differentiation and repertoire diversification of T cells derived from single progenitor cells

Functions of T cells derived from single progenitor cells were investigated. B10. Thy-1.1 recipient mice were either whole body-irradiated and marrow reconstituted or thymus-shielded, irradiated and marrow reconstituted, and limited numbers (3 x 10(3) or 6 x 10(3] of a 1:1 mixture of bone marrow cells from C57BL/6 and B6.Lyt-2.1 mice were transferred intrathymically (i.t.). Donor-type (Thy-1.2+) cells of the thymus of a small portion of recipients which expressed the phenotype of either Ly-2.2 or Ly-2.1 but not both were regarded to be a clone of T cells derived from a single progenitor cell, and such clones were assayed for polyclonal helper (Th) and polyclonal cytolytic (CTL) activities as well as alloantigen-specific proliferative (mixed lymphocyte reaction; MLR) and CTL activities. Clones taken 4 weeks after transfer (4-week-old clones) which were generated in the thymus of whole body-irradiated recipients showed polyclonal CTL but not polyclonal Th activity, whereas 4-week-old clones generated in the thymus of thymus-shielded recipients showed both polyclonal CTL and Th activities. Similarly, 4-week-old clones generated in whole body-irradiated recipients responded with CTL to alloantigens when induced in the presence of T cell growth factors but not with MLR, whereas 4-week-old clones generated in thymus-shielded recipients showed both MLR and CTL to alloantigens. Repertoire diversification of 4-week-old clones, however, was incomplete, since clones generated in whole body-irradiated recipient did not necessarily respond with CTL to all alloantigens examined, and those generated in thymus-shielded recipients did not necessarily respond with MLR to all the antigens. On the other hand, T cell clones were shown to fully mature by 7 weeks after transfer in terms of cell function as well as repertoire diversification.     

Green fluorescent protein-transgenic mice: immune functions and their application to studies of lymphocyte development

Green fluorescent protein (GFP) transgenic (GFP⁺) mice express GFP in most tissues except erythrocytes and hair. Immune responses of GFP⁺ mouse and their application to studies of lymphocyte development were investigated. Flow cytometric analyses revealed that differentiation patterns of lymphocytes from GFP⁺ mice are equivalent to those from parental C57BL/6 mice. There was no difference in mature T-cell proliferative ability in response to allogeneic stimulator cells or anti-CD3 stimulation between GFP⁺ and C57BL/6 mice. Furthermore, the anti-OVA antibody response of GFP⁺ mice was also the same as that of C57BL/6 mice. Taken together, these results show no immunological differences between GFP⁺ and C57BL/6 mice. Bone marrow transplantation and in vitro thymus reconstitution experiments were performed in an attempt to apply the GFP⁺ mice to the analysis of lymphocyte development. When bone marrow cells from GFP⁺ mice were transplanted, T and B lymphocytes containing GFP developed normally in scid recipients. Next we examined intrathymic T-cell development by hanging drop culture methods. GFP⁺ and CD4⁺8⁺ immature T-cells developed normally from bone marrow cells in the reconstituted thymus. The experimental system using hematopoietic cells from GFP⁺ mice is a powerful tool for visualizing lymphocyte development.     

Selection of phenotypically distinct NK1.1+ T cells upon antigen expression in the thymus or in the liver.

Unlike the main TCR alphabeta T cell lineage in which deletion occurs at the CD4+ CD8+ double-positive (DP) stage upon TCR engagement by antigen in the thymus, some T cells appear to require such engagement for their selection, either in the thymus or extrathymically. We used a transgenic TCR (tgTCR) model which, as we previously showed, led to selection upon expression of the corresponding antigen H-2Kb (Kb) in the thymus, of tgTCR/CD3(lo) CD4- CD8- double-negative (DN) thymocytes that expressed the NK1.1 marker (NK T cells) (Curnow, S. J., et al., Immunity 1995. 3: 427). We now report that antigen expression on medullary epithelial cells of the thymus failed to select the NK T cells, whereas its expression on thymocytes did, although tgTCR DP thymocyte development was affected under both conditions. Antigen expression on hepatocytes (Alb-Kb mice) did not perturb tgTCR DP thymocyte development. No enrichment in tgTCR NK T cells was detected in the periphery, except for the liver of the Alb-Kb/tgTCR mice. When reconstitution of thymectomized and irradiated H-2k hosts expressing or not Kb was performed with bone marrow from tgTCR H-2k mice, an enrichment in tgTCR+ NK T cells was found in the liver, but not in the spleen, of the hosts which expressed Kb, either selectively on hepatocytes or ubiquitously. Surprisingly, the majority of the hepatic tgTCR+ NK T cells also expressed the CD8 alpha/beta heterodimer. These results indicate that thymus-independent NK T cells with unique phenotypic characteristics can be selected upon antigen encounter in the liver.     

Inhibition of natural killer cell-mediated lysis of tumor cells by normal and regenerating bone marrow

The natural killer (NK) cells which can lyse certain tumor cells during brief incubation in vitro have also been postulated to be the cells responsible for natural resistance to transplanted hemopoietic cells in vivo. To test this hypothesis, we have now measured: 1) the ability of bone marrow cells to compete with tumor cells as targets for spleen NK cells and 2) the effect of a brief incubation with spleen cells on the hemopoietic grafting potential of bone marrow cells. Firstly, when CBA/J mouse spleen cells were incubated with 51Cr-labelled YAC tumor cells together with DBA/2 mouse bone marrow cells, tumor cell lysis was reduced compared with incubation of spleen cells with tumor cells alone. Tumor cell lysis was even less when post-irradiation regenerating bone marrow was used. Secondly, C57B1/6 mouse bone marrow cells incubated with an excess of DBA/2 mouse spleen cells showed a reduced ability to produce hemopoietic spleen colonies in irradiated 129/J mice, whereas incubation with either thymus cells or fewer spleen cells produced no such effect. The results show that, when incubated with spleen cells under the conditions of a standard NK cell assay, regenerating bone marrow cells competitively inhibit the killing of YAC tumor cells and bone marrow progenitor cells are rendered ineffective in their hemopoietic colony-forming potential (CFU-s). These findings suggest that certain hemopoietic progenitor cells and YAC tumor cells can both serve as targets for NK cells, consistent with the view that the spontaneous cytolysis of tumor cells in vitro and natural resistance to bone marrow transplantation in vivo are mediated by cells of a common lineage.     

Susceptibility of radiation chimeras to Trypanosoma cruzi.

Reciprocal bone marrow transfers were performed with C3H/HeJ mice, which are susceptible to infection with the Brazil strain of Trypanosoma cruzi, and resistant F1 (C3H/HeJ X C57BL/6J) mice. Mice reconstituted after lethal irradiation with syngeneic bone marrow displayed the resistance phenotype of the strain used, but neither C3H mice reconstituted with F1 bone marrow cells nor F1 mice reconstituted with C3H bone marrow cells survived challenge. Resistance to T. cruzi appears to be dependent upon factors associated both with host background and with bone marrow-derived cells.     

Environmental and allele-specific influences on T cell receptor gene rearrangement: skewed alpha, delta, and gamma gene rearrangement patterns in chimeric mice.

This study was performed as a means to examine the effects of environment and stem cell origin on the molding of T cell receptor gene rearrangements during T cell development. Lethally irradiated adult mice were first reconstituted with (C57BL/6 x DBA/2)F1 stem cells, which derived either from a fetal liver or adult bone marrow source. The gene rearrangements in the irradiated thymi were then compared to those in normal fetal or adult thymi by hybridoma analysis. Results showed a general absence of gene rearrangements typical of the normal fetal thymus in both sets of chimeras. alpha and gamma gene rearrangements in chimeric mice matched normal adult patterns. However, chimeric thymocytes were unique in an unusually low frequency of rearrangements at the delta locus. The use of the bone marrow vs. fetal liver as stem cell sources in chimeric mice did not affect the patterns, indicating that environmental factors played a major role in the molding of gene rearrangement at multiple T cell receptor loci. Interestingly, the chromosomal sequence added an additional influence as homologous chromosomes showed allele-specific rearrangement patterns. We now question whether the unique patterns of rearrangement in irradiated adult thymi may substantially affect developing T cell populations in chimeric animals, particularly with regard to the gamma/delta T cell subset. Further analyses are warranted, both in experimental and clinical settings, regarding ultimate potentials for T cell diversity and function in transplantation recipients.