Abrogation of hybrid resistance to bone marrow engraftment by graft-vs-host-induced immune deficiency

Lethally irradiated F1 mice, heterozygous at the hematopoietic histocompatibility locus Hh-1, which is linked with H-2Db, reject bone marrow grafts from H-2b parents. This hybrid resistance (HR) is reduced by prior injection of H-2b parental spleen cells. Because injection of parental spleen cells produces a profound suppression of F1 immune functions, we investigated whether parental-induced abrogation of HR was due to graft-vs-host-induced immune deficiency (GVHID). HR was assessed by quantifying engraftment of H-2b bone marrow in F1 mice with the use of splenic [125I]IUdR uptake; GVHID, by the ability of F1 spleen cells to generate cytotoxic T lymphocytes (CTL) in vitro. We observed a correlation in the time course and spleen cell dose dependence between loss of HR and GVHID. Both GVHID and loss of HR were dependent on injection of parental T cells; nude or T-depleted spleen cells were ineffective. The injection of B10 recombinant congenic spleens into (B10 X B10.A)F1 mice, before grafting with B10 marrow, demonstrated that only those disparities in major histocompatibility antigens that generated GVH would result in loss of HR. Thus, spleens from (B10 X B10.A(2R]F1 mice (Class I disparity only) did not induce GVHID or affect HR, whereas (B10 X B10.A(5R))F1 spleens (Class I and II disparity) abrogated CTL generation and HR completely. GVHID produced by a class II only disparity, as in (B10 X B10.A(5R))F1 spleens injected into (B6bm12 X B10.A(5R))F1 mice, was also sufficient to markedly reduce HR to B10 bone marrow. This evidence that GVHID can modulate hematopoietic graft rejection may be relevant to the mechanisms of natural resistance to marrow grafts in man.     

Induction of unresponsiveness to bone marrow grafts.

Unresponsiveness to Hh incompatible bone marrow grafts was induced in mice by single or multiple injections of various tissues from a prospective donor before irradiation and bone marrow grafting. The results show that lymph node cells and splenocytes (both adherent and nonadherent) were the most effective in inducing unresponsiveness; thymocytes showed only a marginal effect in female and no effect in male mice, and hepatocytes had no effect. There was a direct relationship between the number of cells required for unresponsiveness induction and the strength of incompatibility between donor and recipient, i.e., the stronger the donor-recipient incompatibility, the more cells were required to induce unresponsiveness. The rapidity of unresponsiveness induction and its duration were also dependent on the number of cells in the "immunizing" inoculum. In general, unresponsiveness was induced sooner and persisted longer when larger cell doses were used. The unresponsiveness was highly specific with regard to donor antigens.     

Genetically determined resistance to foreign bone marrow transplantation in mice: characterization of the effector cells

Mice of most strains show a genetically determined ability to reject a variety of foreign marrow grafts even after lethal irradiation. The phenomenon is both host strain and donor marrow graft-dependent. To characterize the effector cell responsible for graft rejection, attempts were made to 1) determine to what morphologic subclass it belongs; 2) determine its life span; and 3) establish whether genetically different host environments influence the functioning of the effector cell. Mice of the 129/J strain (normally nonresistant), C57BL/6 strain (made non-resistant), and the homozygous mutants of C57BL/6, i.e., C57BL/6 (bg/bg), were recipients of C57BL/6 marrow or spleen cells. After lethal irradiation, hosts were given marrow or spleen cells from normal, strongly resistant C57BL/6 donors pretreated with a) 950 R whole body irradiation or b) twice daily injections for 4 days of the cell cycle toxic drug hydroxyurea followed by 950 R. In other cases, hosts were recipients of the lymphoid cell-rich fraction of marrow from irradiated C57BL/6 donors or adherent cells taken from cultures of marrow cells of unirradiated C57BL/6 donors. Three hours after receiving C57BL/6 marrow or spleen cells, irradiated hosts were given allogeneic DBA/2 marrow (always strongly rejected by C57BL/6 mice and always accepted by 129/J strain mice). Seven days later, host spleens were removed and the numbers of microscopic colonies were counted from subserial sections. The results demonstrate that 1) mice either normally or rendered nonresistant to a marrow allograft can be made to develop resistance by the administration of either whole spleen cells or marrow lymphoid cells from lethally irradiated strongly resistant donors; 2) adherent cells from cultures of marrow from strongly resistant mice are ineffective in conferring resistance; 3) the cell effective in conferring resistance has a life span greater than 4 but less than 7 days; and 4) the effector cell can function in genetically different environments of nonresistant strains.     

Stimulation of genetic resistance to marrow grafts in mice by interferon-alpha/beta

Lethally irradiated mice were infused with syngeneic, H-2 allogeneic, parental strain, or H-2 heterozygous bone marrow cells. They were injected daily with rabbit anti-mouse interferons (IFN)-alpha/beta or gamma or with IFN-alpha/beta. The growth of donor-derived cells was judged 5 days later by measuring splenic incorporation of 5-iodo-2'-deoxyuridine-125I into DNA. Antibodies to IFN-alpha/beta, but not to IFN-gamma, weakened genetic (both hybrid and allogeneic) resistance to marrow cell grafts. IFN-alpha/beta stimulated hybrid and allogeneic resistance, the latter even in genetically "poor responder" mice. Mice pretreated with silica, which weakens genetic resistance, were stimulated by IFN-alpha/beta to resist incompatible marrow cell grafts; however, IFN-alpha/beta failed to reverse the effects of antiasialo GM1 serum on marrow graft rejection. IFN-alpha/beta did not inhibit the growth of syngeneic marrow cells and did not stimulate resistance to H-2 heterozygous bone marrow cells. We propose that genetic resistance occurs in two discrete steps. In the first step, hemopoietic histocompatibility (Hh) antigens are recognized by one host cell type, and this recognition leads to IFN-alpha/beta secretion by a silica-sensitive cell. In the second step, asialo GM1-positive natural killer cells stimulated by IFN-alpha/beta recognize Hh antigens on marrow stem cells and cause rejection. The defects in resistance observed in genetically poor responder mice and in mice treated with silica appear to involve the first step in recognition. The lack of rejection of H-2 heterozygous (Hh-) marrow cells by parental strain mice injected with IFN-alpha/beta indicated that specific Hh recognition is critical in the second step of genetic resistance.     

Bone formation by revascularized periosteal and bone grafts, compared with traditional bone grafts.

Three groups of dogs were studied to compare the bone formation obtained with traditional bone grafting techniques, and that obtained with revascularized periosteum or revascularized bone grafts--all in unstressed bone. Revascularized periosteum did not produce a significant amount of bone in this unstressed model. At 4 months the revascularized rib grafts had a slightly greater tolerance to stress at the callus site than traditional bone grafts, but the incidence of non-union was the same. We conclude that the use of revascularized bone grafts should be reserved for situations in which traditional bone grafting techniques are unlikely to be successful.     

Tumor-host cell hybrids in radiochimeras reconstituted with bone marrow and thymus grafts.

CBA/H and CBA/HT676 radiation chimeras were prepared by lethal irradiation and subsequent reconstitution with bone marrow of the host karyotype and thymocytes of the opposite karyotype. After T- and B-cell chimerism had been established, the animals were inoculated with SEWA or TA3Ha ascites tumor--donor cell hybrids were isolated following the explantation of TA3Ha tumor in selective HAT medium and by selecting for adherent cells from SEWA. The T6T6 chromosomal marker served to distinguish between the type of cell involved in the fusion. In all hybrids the donor component was a nonthymus-derived cell.     

The immunoregulatory role of bone marrow. I. Suppression of the induction of antibody responses to T-dependent and T-independent antigens by cells in the bone marrow.

Bone marrow cells (BMC) from normal mice suppressed the in vitro IgM, but not the IgG, antibody (Ab) response of spleen cells. BMC were inhibitory only when added during the first 24 hr of culture, and inhibition was not due to an induced shift in the kinetics of the response. Addition of specifically activated T cells or nonspecific T-cell-replacing factors to normal or T-depleted spleen cell cultures did not abrogate suppression while the response to the T-independent antigen DNP-polymerized flagellin or lipopolysaccharide was also suppressed. BMC did not inhibit background Ab synthesis by normal or primed cells in the absence of antigen and did not inhibit, but stimulated, DNA synthesis in normal spleen cell cultures. In addition, high-avidity Ab synthesis was preferentially suppressed. A possible role for the bone marrow suppressor cell in the induction of B cell tolerance is discussed.     

Abrogation of resistance to the reinduction of experimental allergic encephalomyelitis by pertussigen

Experimental allergic encephalomyelitis is an autoimmune disease initiated by an injection of myelin basic protein in complete Freund's adjuvant. Lewis rats which have recovered from the initial episode of hindquarter paralysis are resistant for at least 6 months to disease reinduction by basic protein-complete Freund's adjuvant, although specific antigen-reactive cells are detectable in convalescent rats. Resistance cannot be attributed to the activity of the adjuvant alone. In contrast, clinical disease could be reinduced by a secondary challenge with spinal cord homogenate and pertussigen (“lymphocytosis promoting factor” of Bordetella pertussis). Disease could also be reinduced by a simultaneous secondary challenge with basic protein-complete Freund's adjuvant along with pertussigen. Vascular permeability increases in the spinal cord paralleled disease induction or reinduction. No definite conclusions can be drawn concerning the mechanism by which pertussigen promotes disease reinduction in convalescent rats.     

Studies on colony formation in vitro by mouse bone marrow cells. I. Continuous cluster formation and relation of clusters to colonies

Colony formation in vitro by mouse bone marrow cells following stimulation by human urine was analysed over a 7-day incubation period. There was a linear increase with time in the number of cell aggregates (clusters) developing in such plates. Early in the incubation period all clusters were granulocytic although later macrophage clusters developed. Although most fully developed colonies were composed of macrophages, mapping and transfer studies showed that at least half of these had initially arisen early in the incubation period as granulocytic clusters.     

Mapping of quantitative trait loci determining NK cell-mediated resistance to MHC class I-deficient bone marrow grafts in perforin-deficient mice

NK cells reject allogeneic and MHC class I-deficient bone marrow (BM) grafts in vivo. The mechanisms used by NK cells to mediate this rejection are not yet thoroughly characterized. Although perforin plays a major role, perforin-independent mechanisms are involved as well. C57BL/6 mice deficient in perforin (B6 perforin knockout (PKO)) reject class I-deficient TAP-1 KO BM cells as efficiently as normal B6 mice. In contrast, perforin-deficient 129S6/SvEvTac mice (129 PKO) cannot mediate this rejection while normal 129 mice efficiently reject. This suggests that in 129, but not in B6, mice, perforin is crucial for NK cell-mediated rejection of MHC class I-deficient BM grafts. To identify loci linked to BM rejection in perforin-deficient mice, we generated backcross 1 progeny by crossing (129 x B6)F(1) PKO mice to 129 PKO mice. In transplantation experiments, >350 backcross 1 progeny were analyzed and displayed a great variation in ability to reject TAP-1 KO BM grafts. PCR-based microsatellite mapping identified four quantitative trait loci (QTL) on chromosomes 2, 4, and 8, with the QTL on chromosome 8 showing the highest significance, as well as a fifth epistatic QTL on chromosome 3. This study describes the first important step toward identifying BM graft resistance gene(s).     

Different sensitivities to hydrocortisone of natural killer cell activity and hybrid resistance to parental marrow grafts.

Natural killer cells and cells mediating F1 anti-parent responses in vivo and in vitro differ in their sensitivity to hydrocortisone acetate. NK cell activity is sharply decreased after in vivo drug administration whereas induction of specific F1 anti-parent or anti-allogeneic cytotoxicity and hybrid resistance to parental marrow grafts are not impaired. Because of several other properties shared by the NK and anti-Hh host reactivities, it is still suggested that the effector cells are generated from a single differentiation pathway, but that they differ with respect to specificity and sensitivity to hydrocortisone.     

Effects of carrageenan on immune responses. Studies on the macrophage dependency of various antigens after treatment with carrageenan.

Carrageenan, a sulfated polygalactose having macrophage toxic properties, elicited a marked suppression of IgM response to T cell-dependent antigens such as sheep red blood cells (SRBC), dinitrophenylated bovine serum gamma-globulin (DNP-BGG), and trinitrophenylated concanavalin A (TNP-Con A). In contrast, carrageenan did not inhibit antibody responses to such T cell-independent antigens as trinitrophenylated DEAE-dextran (TNP-DEAE-dextran), trinitrophenylated polyvinyl pyrrolidone(TNP-PVP), and trinitrophenylated Ficoll (TNP-Ficoll). Compared to total spleen cells, spleen cells from which macrophages had been removed by adhesion to plastic Petri dishes had less effect on the production of antibody against T cell-dependent antigens, but no change or a rather stimulated effect was observed in in vitro antibiody synthesis against T cell-independent antigens. These results strongly suggest that macrophages are involved in antibody responses to T cell-dependent antigens but not in those to T cell-independent antigens. However, the antibody response to trinitrophenylated lipopolysaccharide (TNP-LPS), a T cell-independent antigen, was inhibited by carrageenan treatment, suggesting that the response is macrophage dependent. Moreover, antibody response to higher doses of dinitrophenylated phytohemagglutinin (DNP-PHA), a T cell-dependent antigen, was shown to be macrophage independent by carrageenan treatment, although the antibody response to low doses of the antigen was macrophage dependent. Considering all these results, carrageenan treatment seems to be a very useful method to determine whether immune response to various antigens are macrophage dependent or not.