Alloantigen recognition is critical for CD8 T cell-mediated graft anti-tumor activity against murine BCL1 lymphoma after myeloablative bone marrow transplantation

The goal of the current study was to determine whether whole bone marrow cells or splenic CD8(+) T cells from C57BL/6 (H-2(b)) donor mice, which are tolerant to BALB/c (H-2(d)) alloantigens, are capable of mediating graft anti-tumor activity against a BALB/c B-cell lymphoma after injection into irradiated BALB/c hosts. The experimental results show that high doses of splenic CD8(+) T cells mixed with T cell-depleted bone marrow cells from C57BL/6 non-tolerant (normal) donors eliminate the BCL(1) B-cell lymphoma cells and induce lethal graft-versus-host disease (GVHD). CD8(+) T cells from tolerant donors simultaneously lose both their ability to induce GVHD and their anti-tumor activity. Whole bone marrow cell transplants from normal donors eliminated BCL(1) tumor cells without inducing GVHD, and bone marrow cells from tolerant donors failed to eliminate the tumor cells. The infused BCL(1) tumor cells expressed an immunogenic tumor-specific idiotype antigen disparate from host alloantigens, indicating that recognition of the tumor-specific antigen alone was insufficient to elicit graft anti-tumor activity from unimmunized allotolerant donor splenic CD8(+) T cells or whole bone marrow cells. We conclude that CD8(+) T cells from unimmunized normal donor mice require alloantigen recognition to mediate their anti-tumor activity following allogeneic BMT.     

Anti-third-party veto CTLs overcome rejection of hematopoietic allografts: synergism with rapamycin and BM cell dose

Several bone marrow cells and lymphocyte subpopulations, known as "veto cells," were shown to induce transplantation tolerance across major histocompatibility antigens. Some of the most potent veto cells are of T-cell origin, and in particular a very strong veto activity was documented for cytotoxic T-lymphocyte (CTL) lines or clones. However, these cells also possess marked graft-versus-host (GVH) reactivity. In the present study we evaluated a new approach to deplete CTLs of antihost clones by stimulating the donor T cells against third-party stimulators in the absence of exogenous interleukin 2 (IL-2). We demonstrate that such CTLs are depleted of GVH reactivity while maintaining marked veto activity in vitro. Furthermore, marked synergism was exhibited between the veto CTLs and rapamycin when tested in a murine model, which measures T-cell-mediated bone marrow allograft rejection, or in sublethally irradiated allogeneic hosts. Our results suggest that engraftment of early progenitors could be enhanced by using host-nonreactive anti-third-party CTLs, in conjunction with nonmyeloablative rapamycin-based conditioning protocols, thereby significantly reducing the toxicity of allogeneic transplantation.     

The factors controlling stem cell recirculation. i. Migration of hemopoietic stem cells in adrenalectomized mice

The influence of bilateral adrenalectomy on hemopoietic stem cell (CFU) migration in mice has been studied. Formation of endogenous spleen colonies in lethally irradiated, leg-shielded mice was sharply increased by prior adrenalectomy, and this increase was not dependent on the volume of shielded bone marrow. Adrenalectomy was shown to increase endogenous spleen colony formation in sublethally irradiated mice as well. However, it had no affect on formation of spleen colonies in lethally irradiated mice injected with syngeneic bone marrow. The CFU content of murine bone marrow decreased acutely after removal of the adrenals, and this decrease was accompanied by a concomitant increase in the peripheral blood and splenic CFU. Thus, adrenalectomy appeared to have no affect on the splenic plating efficiency or proliferative rate of hemopoietic stem cells, but it did result in increased migration of stem cells from the bone marrow to the blood, and thence to the spleen. It is concluded that the adrenal steroids may be of physiologic importance in the regulation of ehmopoietic stem cell migration.     

Transfer of immunity by transfer of bone marrow cells: a requirement for T lymphocytes and sensitivity to cyclophosphamide.

Thymectomized, lethally irradiated mice reconstituted with bone marrow cells (TIR mice) from normal donors succumbed after i.p. challenge with xenogeneic (rat) Yoshida ascites sarcoma (YAS) given 1 month after irradiation and reconstitution. YAS rejection and production of YAS-antibodies was induced in TIR mice by a single i.v. injection of normal syngeneic spleen cells given 1 day after the tumor. Purified splenic T lymphocytes also induced YAS rejection in TIR mice, but splenic B lymphocytes did not affect progressive tumor growth. Tumor was also rejected in TIR mice that had been reconstituted with bone marrow cells from YAS-immune donors. The sera of these TIR mice did not contain tumor antibodies between reconstitution and YAS challenge, but a high YAS-antibody titer was present after YAS challenge and rejection. Immunofluorescence did not reveal any dramatic differences in the spleen and bone marrow content of T and B lymphocytes of TIR mice reconstituted with cells from normal donors and those reconstituted with cells from the YAS-immunized donors. Transfer of YAS-resistance was abolished when the bone marrow cells from immunized donors were treated with Thy 1.2 antiserum and complement, or when bone marrow donors were injected with cyclophosphamide 1 day after immunization. Cyclophosphamide was also shown to induce strong and specific suppression of YAS-antibody production in normal mice.     

Transfer of colitis by Galphai2-deficient T lymphocytes: impact of subpopulations and tissue origin

To elucidate the potential cell population(s) involved in the induction of colitis in inhibitory G protein Galphai2(-/-) mice, Galphai2-deficient or competent bone marrow or splenic and mesenteric lymph node (MLN) T cells were transferred into immunodeficient mice. The mice were followed up to 23 weeks after transfer, recording changes in body weight. Colitis was graded on hematoxylin and eosin-stained colonic tissue, and production of serum interleukin-18 and colon-derived interferon-gamma was measured using ELISA. After adoptive transfer of Galphai2(-/-) bone marrow, severe colitis developed in irradiated wild type recipients, whereas irradiated Galphai2(-/-) mice increased their life span more than 3 times after transfer of wild type bone marrow, accompanied by significant amelioration of colitis. Neither purified Galphai2(-/-) CD4(+), nor CD8(+) splenic or MLN-derived T cells could induce colitis in recombination-activating gene V(RAG) 2(-/-) recipient mice, whereas transfer of splenic Galphai2(-/-) CD3(+) T cells induced severe colitis. In contrast, transfer of Galphai2(-/-) CD3(+) T cells from the MLN caused only minor histopathological changes in the intestinal mucosa. Finally, serum levels of interleukin-18 and interferon-gamma production from colonic tissue cultures correlated well with disease severity. Our results show that bone marrow transplantation can prolong the life of Galphai2(-/-) mice and ameliorate intestinal inflammation. Splenic CD4(+) or CD8(+) T cells on their own were poor inducers of colitis, whereas the combination of both was highly involved in the induction of intestinal inflammation. Furthermore, we show that the tissue origin of CD3(+) T cells is critical for their potency to induce colitis.     

Protection from lethal murine graft-versus-host disease without compromise of alloengraftment using transgenic donor T cells expressing a thymidine kinase suicide gene

Donor T cells play a pivotal role in facilitating alloengraftment but also cause graft-versus-host disease (GVHD). Ex vivo T-cell depletion (TCD) of donor marrow is the most effective strategy for reducing GVHD but can compromise engraftment. This study examined an approach whereby donor T cells are selectively eliminated in vivo after transplantation using transgenic mice in which a thymidine kinase (TK) suicide gene is targeted to the T cell using a CD3 promoter/enhancer construct. Lethally irradiated B10.BR mice transplanted with major histocompatibility complex (MHC)-incompatible TCD C57BL/6 (B6) bone marrow (BM) plus TK(+) T cells were protected from GVHD after treatment with ganciclovir (GCV) in a schedule-dependent fashion. To examine the effect of GCV treatment on alloengraftment, sublethally irradiated AKR mice underwent transplantation with TCD B6 BM plus limiting numbers (5 x 10(5)) of B6 TK(+) T cells. Animals treated with GCV had comparable donor engraftment but significantly reduced GVHD when compared with untreated mice. These mice also had a significantly increased number of donor splenic T cells when assessed 4 weeks after bone marrow transplantation. Thus, the administration of GCV did not render recipients T-cell deficient, but rather enhanced lymphocyte recovery. Adoptive transfer of spleen cells from GCV-treated chimeric mice into secondary AKR recipients failed to cause GVHD indicating that donor T cells were tolerant of recipient alloantigens. These studies demonstrate that administration of TK gene-modified donor T cells can be used as an approach to mitigate GVHD without compromising alloengraftment.     

Correction of X-linked immunodeficient mice by competitive reconstitution with limiting numbers of normal bone marrow cells.

Gene therapy for inherited disorders is more likely to succeed if gene-corrected cells have a proliferative or survival advantage compared with mutant cells. We used a competitive reconstitution model to evaluate the strength of the selective advantage that Btk normal cells have in Btk-deficient xid mice. Whereas 2,500 normal bone marrow cells when mixed with 497,500 xid cells restored serum IgM and IgG3 levels to near normal concentrations in 3 of 5 lethally irradiated mice, 25,000 normal cells mixed with 475,000 xid cells reliably restored serum IgM and IgG3 concentrations and the thymus-independent antibody response in all transplanted mice. Reconstitution was not dependent on lethal irradiation, because sublethally irradiated mice all had elevated serum IgM and IgG3 by 30 weeks postreconstitution when receiving 25,000 normal cells. Furthermore, the xid defect was corrected with as few as 10% of the splenic B cells expressing a normal Btk. When normal donor cells were sorted into B220(+)/CD19(+) committed B cells and B220(-)/CD19(-) cell populations, only the B220(-)/CD19(-) cells provided long-term B-cell reconstitution in sublethally irradiated mice. These findings suggest that even inefficient gene therapy may provide clinical benefit for patients with XLA.     

CXCR4 and CCR5 mediate homing of primitive bone marrow-derived hematopoietic cells to the postnatal thymus

Factors governing the entry of cells into the postnatal thymus are poorly understood. We aimed to define molecular mechanisms mediating the homing of bone marrow cells to the thymus using a sublethally irradiated in vivo murine model. Entry of unfractionated and lineage-depleted bone marrow cells to the thymus, but not bone marrow, was a Galphai-mediated phenomenon. Lineage-depleted cells that had homed to the thymus expressed abundant CXCR4 and CCR5 mRNA, alone of 17 chemokine receptors evaluated by QPCR. Thymic-homed cells were distinct from cells that had homed to bone marrow in expression of CXCR4 and CCR5 by mRNA quantification and cell-surface expression of protein. Abrogation of CXCR4 and CCR5 function by genetic, antibody, or pharmacologic means impaired homing of lineage-depleted cells to the thymus, although not in a synergistic manner, implying interdependency of these receptors in the homing process. Competitive repopulation experiments demonstrated that inhibiting CXCR4-mediated homing adversely affected the double-negative cell pool at 2 weeks, suggesting that cells with prothymocytic activity may in part home via CXCR4. Overall, our data demonstrate differential homing mechanisms governing entry of unfractionated and lineage-depleted cells to irradiated bone marrow or thymus, with thymic homing of immature cells being pertussis-sensitive and mediated by the chemokine receptors CXCR4 and CCR5.     

Hematopoietic reconstitution after lethal irradiation and bone marrow transplantation: effects of different hematopoietic cytokines on the recovery of thymus, spleen and blood cells

Lethally irradiated mice were grafted with syngeneic bone marrow cells or left ungrafted. Mice of each group were injected with different hematopoietic cytokines for 5 consecutive days starting immediately after irradiation or left uninjected. The recovery of lymphoid tissues induced by hematopoietic cytokines 7 days after irradiation and bone marrow cell transplantation was comparable to that observed at days 21-28 in irradiated, bone marrow-grafted, but cytokine-uninjected mice. IL-11 or IL-6, in combination with IL-3, was able to hasten thymus, spleen and blood cell numbers and functions. SCF also displayed a detectable effect when used with IL-3. Conversely, the IL-6 superagonist K-7/D-6 was able, when injected alone, to induce significant recovery of thymus, spleen and blood cells. Thus, K-7/D-6 appears to be a most efficient cytokine for fast reconstitution of lymphoid tissues after irradiation and bone marrow transplantation.     

Pattern of localization of primitive hematopoietic cells in vivo using a novel mouse model.

Bone marrow transplantation is increasingly used as a treatment for numerous immunologic, hematologic, and malignant disorders. However, the mechanism by which transplanted hematopoietic stem cells are engrafted is not completely understood. Many traditional techniques have been used to study the engraftment of transplanted stem cells. Most of these methods are ex vivo and, in some cases, donor cells must be modified to enable detection. We describe a novel alternative for identifying unmodified primitive donor cells in a murine host. This mouse model is based on the differential capacity of adenine phosphoribosyltransferase (APRT)-positive and APRT-negative cells to sequester and incorporate radiolabeled adenine. Aprt is the gene encoding the adenine phosphoribosyltransferase purine salvage enzyme and has been ablated in 129sv mice. Following the injection of APRT-positive c-kit-positive enriched hematopoietic cells into syngeneic, sublethally irradiated APRT-deficient mice, engrafted cells and their presumptive progeny were successfully tracked by polymerase chain reaction. Their presence also was visualized by autoradiography of paraffin-embedded tissue sections. APRT-positive c-kit-positive enriched cells were detected in the bone marrow, spleen, lung, and thymus of nonirradiated mice. Donor cells and their progeny were more widely distributed in tissues of sublethally irradiated mice than of their nonirradiated counterparts, demonstrating that the pattern of localization of c-kit-positive enriched cells differs between nonirradiated and sublethally irradiated syngeneic recipients. The Aprt mouse model provides a sensitive method for further studying the mechanism of engraftment of unmodified donor hematopoietic cells in relation to the tissue architecture of the recipient.     

Age-dependent appearance of non-major histocompatibility complex-restricted helper T cells.

T cells which recognize antigen in association with self major histocompatibility complex (MHC) molecules are positively selected within the thymus. This results in skewing of the T-cell repertoire toward the recognition of antigenic peptides presented by self MHC molecules. While the thymus gland involutes at a relatively young age, bone marrow function and the size of the peripheral T-cell pool are maintained with age. We have investigated the MHC restriction of helper T-cell function for B-cell production of specific antibody in mice of different ages. Splenic helper T cells from 2- to 3-month old mice immunized with phosphocholine-keyhole limpet hemocyanin conjugate were MHC-restricted as defined by their capacity to induce phosphocholine-specific antibody synthesis by syngeneic but not by allogeneic B cells. In contrast, splenic T cells from immunized 18- to 20-month-old mice induced specific anti-phosphocholine antibodies by both syngeneic and allogeneic B cells. No evidence of polyclonal immunoglobulin synthesis was detected. The ability of T cells from old mice immunized with phosphocholine-keyhole limpet hemocyanin to induce phosphocholine-specific antibody synthesis by B cells from allogeneic mice was inhibited by T cells from immunized young mice. These findings suggest that non-MHC-restricted T-cell helper activity in old mice results from the loss of T cells, present in young mice, which suppress non-MHC-restricted helper cells.     

Enhanced antibody affinity in sublethally irradiated mice and bone marrow chimeras.

Sublethally irradiated mice primed with dinitrophenyl (Dnp)-keyhole limpet hemocyanin immediately after irradiation or 30 days later and subsequently boosted with a second injection of antigen displayed a secondary response to Dnp characterized by antibody affinity greater than that in unirradiated controls. Also, in radiation chimeras primed with Dnp-keyhole limpet hemocyanin 120 days after syngeneic or allogeneic bone marrow transplantation the antibodies against Dnp produced after boosting were of higher affinity than the antibodies raised in normal mice. These findings are tentatively attributed to lack of suppressor thymus-derived lymphocytes (T cells) in sublethally irradiated mice and bone marrow chimeras, in which the enhanced ability to produce antibodies of high affinity may compensate for quantitative defects of the immune system.     

Defective in vitro migratory capacity of bone marrow cells from viable motheaten mice in response to normal thymus culture supernatants

"Viable motheaten" mice are severely immunodeficient and develop autoantibodies early in life. The thymus appears normal for the first 3-4 weeks, after which there is depletion of cortical thymocytes and a diminution in the size of the organ until it is atrophic. The present study utilized an in vitro migration assay, in which bone marrow cells from viable motheaten mice were found to have a greatly diminished capacity to migrate in response to normal thymus supernatant when compared to normal bone marrow cells. It was also determined that thymus supernatant prepared from newborn viable motheaten mice was chemoattractive to normal bone marrow but not to viable motheaten bone marrow. The results of in vivo reconstitution of lethally irradiated viable motheaten mice with normal bone marrow cells also show that the thymus of the mutant is normal in its ability to attract and be repopulated by normal donor bone marrow. Therefore, the premature thymic involution of viable motheaten mice is related to the inability of bone marrow cells from these mice to migrate or respond to signals from the thymus.     

Differential effects of dehydroepiandrosterone (DHEA) on murine lymphopoiesis and myelopoiesis.

Dehydroepiandrosterone (DHEA) inhibits murine lymphopoiesis, especially after sublethal irradiation, without affecting mature lymphocyte function. We demonstrate here that dietary DHEA differentially affected myelopoiesis and lymphopoiesis in sublethally irradiated mice. Erythropoietic progenitor cell and stem cell function was not affected by DHEA although the magnitude of granulopoiesis was slightly reduced. Regeneration of marrow B220+ B cells, natural killer (NK) function, and thymus repopulation were significantly delayed in DHEA-fed mice. Interleukin 2 (IL-2) failed to restore NK activity of DHEA-fed mice to normal levels. Marrow from DHEA-fed mice contained competent thymocyte progenitors capable of repopulating thymi of irradiated hosts fed a control diet but not those fed the DHEA diet. Thus DHEA inhibits lymphopoiesis while sparing myelopoiesis, affecting the growth and maturation of T, B, and NK cells by a mechanism other than the inhibition of IL-2 production.     

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.     

Inflammatory arthropathy in MRL hematopoietic chimeras undergoing Fas mediated graft-versus-host syndrome

OBJECTIVE: To determine whether overexpression of the Fas ligand (FasL) on activated lpr T lymphocytes could induce arthritic lesions when grafted into syngeneic wild-type MRL mice expressing normal Fas receptor levels. METHODS: Lethally irradiated MRL+/+ mice were reconstituted with congenic MRL/lpr bone marrow cells and splenocytes overexpressing FasL. Fas-mediated cytotoxic properties of repopulating lpr splenic lymphocytes were evaluated in vitro. Simultaneously, the hind paw ankles of the hematopoietic chimeras were histologically examined. RESULTS: The lpr lymphocytes repopulating the spleen overexpressed FasL and had in vitro Fas-mediated cytotoxic activity. Simultaneously, in vivo, articular (synovitis, pannus) and periarticular (periostitis) inflammation with bone resorption were observed. CONCLUSION: Arthritic lesions may be induced in Fas-expressing recipients by persistent engrafted syngeneic lymphocytes overexpressing FasL.     

Evaluation of TCR Vbeta subfamily T cell expansion in NOD/SCID mice transplanted with human cord blood hematopoietic stem cells

Examination of the T cell receptor (TCR) gene repertoire is important in the analysis of the immune status of models, because clonal expansion of T cells permits the identification of specific antigen responses of T cells. Little is known about T-cell immunity in the humanized NOD/SCID mouse model. TCR Vbeta repertoire usage and clonality were analyzed to investigate the distribution and clonal expansion of TCR Vbeta subfamily T cells in NOD/SCID mice transplanted with human cord blood (CB) hematopoietic stem cells. The NOD/SCID mice were sublethally irradiated ((60)Co, 300cGy) to eliminate residual innate immunity in the host. The experimental mice were transplanted intravenously with CB CD34(+) cells sorted by MACS. After 6 weeks, RNA was obtained from peripheral blood, bone marrow and thymus of the study animals. The gene expression and clonality of the TCR Vbeta repertoire were determined by RT-PCR and GeneScan techniques. A restricted range of TCR Vbeta usage was exhibited in the bone marrow of mice, which included TCR Vbeta 1, 2, 9, 13 and 19. Further, oligoclonal expression of some TCR Vbeta subfamilies (Vbeta9, 13, 19) was identified by GeneScan technique. To investigate the reason for oligoclonal expansion of the TCR Vbeta subfamily T cells from CB in mouse models, the T-cell culture with tissue-antigen of NOD/SCID mouse was performed in vitro. The cells from peripheral blood mononuclear cells and bone marrow, spleen, thymus in NOD/SCID mice were frozen and thawed, and used as tissue-antigen. CB mononuclear cells were separately cultured with the component from those murine cells for 15-20 days. Oligoclonal expression or oligoclonal trend of some TCR Vbeta subfamilies (Vbeta10, 11 and Vbeta2, 15, 16, 19) was detected in T cells after stimulation with tissue-antigen of NOD/SCID mouse. Interestingly, a similar clonal expansion of the TCR Vbeta11 subfamily was found in T cells cultured with peripheral blood, bone marrow and spleen respectively. The TCR Vbeta subfamily T cells could be reconstituted in humanized NOD/SCID mouse transplanted with CD34(+) cells from CB. The restricted expression and clonal expansion of some CB T cell clones may be induced by tissue-antigens of NOD/SCID mice.     

Transfer of experimental antiphospholipid syndrome by bone marrow cell transplantation the importance of the t cell

Objective. To investigate the potential of bone marrow cells from mice with primary antiphospholipid syndrome (APS) to transfer the disease to naive mice, and to determine the importance of the role of T cells in the APS. Methods. Experimental primary APS was induced in naive mice following active immunization with anticardiolipin (aCL) monoclonal antibody (MAb). Whole-population or T cell–depleted bone marrow cells from mice with experimental primary APS were infused into total body–irradiated naive BALB/c recipients. Results. Bone marrow cells (in the presence of T cells) had the potential to induce experimental APS in naive mice, which resulted in high serum titers of aCL, antiphosphatidylserine, and antiphosphatidylinositol antibodies; an increased number of antibody-forming cells specific for each of the above phospholipids; a positive lymph node cell proliferative response to aCL MAb; and clinical features of primary APS, including thrombocytopenia, prolonged activated partial thromboplastin time (indicating the presence of lupus anticoagulant), and a high frequency of fetal resorptions (the equivalent of human fetal loss). T cell–depleted bone marrow cells did not transfer the disease. Conclusion. This study demonstrates the important role of T cells in the development and transfer of experimental primary APS and raises the possibility of T cell manipulations in treatments to prevent this condition.     

Regulatory mechanism of granulopoiesis in the bone marrow of CSF- producing tumor-bearing nude mice

The regulatory mechanism of differentiation of granulocyte and macrophage precursor cells (G/M CFU-C) in bone marrow and spleen obtained from nude mice bearing colony-stimulating factor (CSF) producing tumor (G-mice), which developed marked granulocytosis, was studied. In these mice, granulopoiesis is enhanced in the spleen, but suppressed in bone marrow. Coculture of G-mouse bone marrow cells with splenic cells of control nude mice (C-mice) and of G-mice resulted in 68% and 62%, respectively, more colonies than expected, while coculture of C-mouse bone marrow cells with these two sources of cell fractions resulted in only 2% and 11% more colonies. In the double-layer agar culture system, bone marrow and splenic cells of C- and G-mice produced a maximal number of colonies containing adequate amounts of human urinary CSF in the upper layer when C-mouse bone marrow cells were added to the lower layer, while these four sources of cells produced a moderate or minimal number of colonies when splenic cells of C- and G- mice or G-mouse bone marrow cells were added to the lower layer. Morphological examination of colonies formed in the upper layer revealed that addition of C-mouse bone marrow cells or irradiated G- mouse bone marrow cells to the lower layer resulted in a massive increment in the number of colonies with pure granulocytes and granulocyte and macrophage mixed (G + G/M) colonies formed by G-mouse bone marrow cells in the upper layer. However, the addition of irradiated C-mouse bone marrow cells or G-mouse bone marrow cells before irradiation to the lower layer did not change G + G/M colony formation by G-mouse bone marrow cells in the upper layer. We could reproduce these findings with conditioned media obtained from 3-day liquid cultures of these cell fractions. This suggests that a diffusible factor may be necessary for inhibition of G + G/M colony formation in G-mouse bone marrow cells. The fine mechanism of such inhibition remains to be clarified.     

Marrow transplantation from tolerant donors to treat and prevent autoimmune diseases in BXSB mice.

Autoimmune-prone BXSB male mice were supralethally irradiated and transplanted with CBA/H bone marrow cells. A complete and long-term chimerism was established when donor mice had been induced to develop tolerance of BXSB male antigens by combined treatment with BXSB male spleen cells and cyclophosphamide. Such chimeras did not express autoimmune phenomena or develop lethal autoimmune manifestations. Nor did the recipient mice develop the wasting syndrome or evidence of persistent immunodeficiencies that have been seen in other strains of autoimmune-resistant mice that had been transplanted with bone marrow cells across major histocompatibility complex barriers following an initial purging of the bone marrow of Thy-1+ cells using anti-Thy-1+C.