Evidence that Ia+ bone-marrow-derived cells are the stimulus for the intestinal phase of the murine graft-versus-host reaction

We have investigated whether bone marrow (BM) or tissue-derived cells provide the stimulus for the intestinal phase of graft-versus-host reaction (GVHR) in F1 mice injected with parental spleen cells. After injection of CBA cells, the characteristic increases in crypt length, crypt cell production rate (CCPR), and intraepithelial lymphocyte (IEL) count occurred in the jejunum of (CBA x BALB/c)F1----CBA BM chimeras, but not in CBA----(CBA x BALB/c)F1 BM chimeras. Thus, BM-derived cells alone can induce intestinal GVHR and, as similar intestinal alterations were found in (A.TH x A.TL)----A.TL BM chimeric mice after injection of A.TL spleen cells, we deduce that the BM-derived stimulator cells are Ia+. We conclude that the intestinal pathology in this model of GVHR is an indirect effect of soluble mediators that are released during a local delayed-type hypersensitivity (DTH) reaction induced by recognition of Ia+ passenger leukocytes within the mucosa.     

Factors contributing to the decrease in concanavalin A-induced colony-stimulating factors in acute suppressive graft-versus-host disorder

We have recently demonstrated that IL-3 and GM-CSF content in concanavalin A--stimulated splenic cultures was severely depressed for several weeks in mice undergoing an acute graft-vs.-host reaction (GVHR). Several factors contributed to this decrease in CSF levels at different points in the first 10 weeks of GVH. Myeloid lineage cells, both GM-CFU and Mac-1+ cells, increased in the spleen during GVH weeks 3-6, and (125I)-GM-CSF binding by spleen cells increased 10 fold--hence CSF content in supernatants was probably reduced by local consumption during these weeks. During GVH weeks 3-12, levels of all T cells--and of L3T4+ T cells, in particular--were reduced, limiting production of T cell derived lymphokines. Finally, during the first six weeks of GVH, immune suppression was found in cocultures of normal F1 and GVH spleens. Furthermore, during weeks 3-5, nonspecific suppression was found, affecting CSF production not only by normal host spleens but also by parental donor or unrelated spleen cells as well.     

Lethal graft-versus-host reaction induced by parental cells in the clawed frog, Xenopus laevis

(J X K)F1 hybrid frogs, Xenopus laevis, were produced by mating between the MHC-homozygous but distinct "J" and "K" group frogs. When the spleen or splenocytes from parental J were transplanted into the F1 frogs, a typical graft-versus-host reaction (GVHR) was induced that included wasting and death, accompanied by a prominent spleen enlargement and other histological changes in skin and lymphoid organs. To induce the reaction, sensitization against F1 cells prior to transfer of spleen was necessary. The reaction was accelerated when the parental donor cells had been sensitized by gamma-irradiated leukocytes from the F1 hybrid. The injection of presensitized triploid J splenocytes into F1 hybrids followed by ploidy analyses revealed that the donor cells constituted the major cell population in host lymphoid organs--such as the spleen, liver, and kidney--but not the thymus. These observations provide the first clear evidence in poikilothermic vertebrates for GVHR caused by parental lymphocytes.     

Elimination of self-reactive CD8+, but not CD4+, T cells by a peripheral immune mechanism

Unirradiated (BALB/c X B6)F1 recipients of lymphocytes from either parent or (B6 X DBA/2)F2 recipients of DBA/2 parental lymphocytes specifically remove the function of donor-derived F1-reactive CTL from the spleen, since such cells could not be recovered 1 week after injection. However, donor-derived CTL specific for third-party antigens, as well as donor-derived F1-reactive CD4+ T cells could be recovered. In contrast, CTL in spleens from recipients sublethally irradiated prior to injections consisted predominantly of F1-reactive CTL in all strain combinations tested. Athymic BALB/c nude mice grafted with fetal thymus of B6 develop a T cell compartment tolerant of BALB/c and B6, like (BALB/c X B6)F1 animals. However, unlike the F1 mice, the thymus-grafted nude mice were not able to eliminate B6-reactive lymphocytes after injection of normal BALB/c spleen cells. Our data indicate the existence of a peripheral immune mechanism capable of selectively eliminating self-reactive CD8+ CTL, but not CD4+, T cells. This mechanism requires self antigen expressed on radiosensitive cells. The presence of T cells tolerant to self antigen by thymic negative selection is not sufficient and perhaps not required. Most likely, this mechanism is involved in the relative resistance to lethal GVHR mediated by parental CD8+ T cells in parent-into-F1 situations.     

Altered threshold for the induction of graft-versus-host immunodeficiency following murine cytomegalovirus infection. Host and donor contributions

Acute murine cytomegalovirus (MCMV) infection enhances the ability of parental spleen cells to induce graft-vs.-host immunodeficiency (GVHID) in F1 hybrid mice when the two processes occur simultaneously in the recipient. The present study assessed GVHID as the ability of spleen cells to generate in vitro cytotoxic T lymphocyte responses to trinitrophenyl-modified syngeneic cells. The results indicate that MCMV infection not only reduces the number of parental spleen cells required to induce GVHID, but accelerates the onset of GVHID, which occurs as early as 3 days after cell and virus challenge. To determine whether MCMV infection exerts this synergistic effect primarily through the donor or the host component, we examined the effect of MCMV infection of either donor mice or recipient mice at 3, 10, and 17 days prior to spleen cell transfer. Two weeks after cell transfer, splenocytes were tested for their ability to generate CTL. When donor mice were infected with MCMV three days prior to cell transfer, the ability of donor cells to induce GVHID was reduced. In contrast, MCMV infection of the recipients three days prior to cell transfer increased their susceptibility to GVHID induction. Infection of either donor or host mice 10 days or 17 days prior to parental spleen cell transfer had little effect on the ability to induce or resist GVHID when compared with sham-infected mice. Thus, acute MCMV infection can modulate the severity of GVHID depending on whether it is the donor or the host that is infected. The ability of acute MCMV to alter the course and severity of GVHID may be relevant for human bone marrow transplants in which preceding CMV infection has been associated with chronic GVH. In this setting, CMV may lower the threshold necessary to induce a GVH reaction.     

Disseminated systemic expression of the "local" popliteal lymph node assay in rats.

In contrast to the traditional belief that the popliteal lymph node (PLN) assay of graft-versus-host (GVH) reactivity is a local phenomenon, this study describes significant systemic components of both donor and host lymphoid cell activity. Not only are host radiosensitive lymphocytes of systemic origin necessary for the manifestation of normal PLN hypertrophy, but as few as 2.5 X 10(6) parental lymph node cells (LNCs) injected into the hind footpad of adult F1 hybrid rats disseminate widely, provoking significant systemic GVH reactions, as measured by splenomegaly, and distant lymphadenopathy. Futhermore, locally injected donor LNCs ultimately engender three forms of attenuated GVH reactivity: (1) the dissipation of potentially unlimited GVH reactivity, (2) refractoriness of the host to subsequent rechallenge by GVH-inducing cells, and (3) progressive loss of GVH reactivity in donor LNCs when serially transferred to secondary F1 recipients. Whether this modulation of the cell-mediated immune response is the expression of an anti-recognition structure response by the host or the activation of some other immunoregulatory protein, it is absent or reduced in splenectomized recipients. The data suggest that the spleen provides an immunoregulatory microenvironment in which cell-mediated immune responses, such as the GVH reaction, are modulated.     

Specific resistance to local graft-versus-host reaction in F1 hybrids pretreated intravenously with parent-strain spleen cells. Role of cytotoxic T lymphocytes directed against receptors for the major histocompatibility complex

Pretreatment of B6D2F1 hybrids by injection of B6 spleen cells by subcutaneous or intravenous routes induces specific resistance to the local graft versus host reaction provoked by s.c. engraftment of B6 spleen cells. This resistance has been attributed to the presence in the pretreated F1 hybrids of cytotoxic T lymphocytes directed against receptors that recognize the D2 alloantigens (anti-D2-receptor CTL). However, this hypothesis would seem to be challenged, at least partially, by our previously published results showing that a) when tested before induction of GVHR, the anti-D2-receptor CTL are detectable in F1 hybrids pretreated only by the s.c. route but not by the i.v. route; and b) specific resistance to GVHR observed in i.v.-pretreated F1 hybrids is mediated by a nylon-adherent, Thy-1-, radioresistant (2000 rads) suppressor cell of B6 origin that does not manifest any anti-D2-receptor CTL activity. However, these results did not allow us to exclude the possibility of the presence, in the i.v.-pretreated F1 hybrids, of anti-D2-receptor precursor CTL that could be reactivated during the GVHR by the D2-receptors expressed on the proliferating clone of grafted B6 cells, then differentiate to the receptor-specific CTL effectors that control the development of the GVHR. That is why we have studied in the present work the CTL activity developed against D2-receptors after induction of GVHR in either normal or resistant F1 hybrids. Our results show that F1 hybrids protected against GVHR by i.v. pretreatment with B6 cells or by a transfer of nylon-adherent spleen cells from i.v.-pretreated syngeneic F1 mice do not manifest enhanced anti-D2-receptor CTL activity. When considered along with our previous observations, these results favor our hypothesis that anti-D2-receptor CTL are not involved in the specific resistance to GVHR observed in the i.v.-pretreated F1 hybrids.     

Lymphocyte traffic within the bone marrow and selective retention of alloreactive cells.

Earlier studies showed that large numbers of isotopically labelled thoracic duct lymphocytes (TDLs) enter the bone marrow (BM) within hours of injection but depart equally as rapidly by 12 to 24 hr. The significance of this rapid flux was investigated further. Early (1/2 to 2 hr) after the i.v. injection of TDLs, BM was shown to contain T cells capable of initiating a graft-versus-host (GVH) reaction in F1 hybrids and in other experiments memory cells against human serum albumin (HSA). Both GVH and memory cell activity had markedly declined in the BM by 12 hr. In contrast to the rapid departure of TDLs from syngeneic BM, F1 hybrid BM retained parental lymphocytes with GVH activity for alloantigens of the opposite parent. F1 hybrid BM under these circumstances supported the transformation and proliferation of lymphocytes activated in situ by alloantigens. These selectively retained T cells also reacted to third-party alloantigens. In addition, TDLs with memory for HSA were retained in the BM of F1 hybrids. The BM is a site in which alloreactive immune responses may be initiated or sustained.     

Cytotoxicity in graft-versus-host reaction. II. Lysis of target cells of parental genotyppe by F1 hybrid macrophages.

Graft-versus-host (GVH) reactions were induced in adult F1 hybrid mice with the i.p. injection of parental strain spleen cells. Peritoneal exudate and spleen cells of the F1 hybrids taken 8 days after the induction of GVH reaction had a nonspecific in vitro cytotoxic effect which was measured by using 51Cr-labeled target cells of parental genotype. The cytotoxic cells in the peritoneal exudates were shown to be macrophages which adhered to plastic surfaces and were sensitive to the toxic action of crystalline silica particles. Moreover, the injection of partially purified syngeneic macrophages into the F1 hybrids undergoing GVH reactions increased the cytotoxic activity of the peritoneal exudate cells obtained from these animals. These results suggest that during GVH reaction host macrophages are activated into a state of nonspecific cytotoxicity.     

Specific and nonspecific resistance to local graft-versus-host reaction in F1 hybrids pretreated intravenously with parent-strain spleen cells. I. Two distinct mechanisms

B6D2F1 hybrid mice pretreated i.v. with 5 X 10(7) spleen cells from B6 donors seven days earlier (B6-pretreated B6D2F1 hybrids) develop resistance to local GVHR induced by the subcutaneous injection of spleen cells of either B6 (GVHR-B6) or D2 (GVHR-D2) origin. This resistance has specific and a nonspecific components that concern the GVHR-B6 and the GVHR-D2, respectively. The two types of resistance to GVHR are neither induced under the same conditions nor mediated by the same mechanism. Specific resistance to GVHR is observed in B6D2F1 hybrids pretreated with unseparated, anti-Lyt-1.2+C' treated or 1000 rads-irradiated B6 cells, but not in B6D2F1 hybrids pretreated with anti-Thy-1.2+C' or anti Lyt-2.2+C'-treated B6 cells. In contrast, nonspecific resistance to GVHR is induced only by pretreatment with unseparated B6 cells. Treatment of B6 cells with anti-Thy-1.2, anti-Lyt-1.2, or anti-Lyt-2.2 moAb plus C', or their irradiation at 1000 rads completely abolishes their capacity to induce the nonspecific resistance to GVHR. Moreover, specific resistance to GVHR can be transferred to normal B6D2F1 mice by injection of nylon-adherent, anti-Thy-1.2+C'-treated or 2000-rads-irradiated, but not unseparated or nylon-nonadherent, B6-pretreated B6D2F1 spleen cells. Treatment of nylon-adherent B6-pretreated B6D2F1 cells with anti H-2d antiserum plus C' does not affect their capacity to transfer specific resistance to GVHR. Nonspecific resistance to GVHR can be transferred by unseparated, anti-Lyt-1.1+C' or anti Lyt-2.1+C'-treated, but not by anti-Thy-1.2+C' anti-Lyt-1.2+C', anti-Lyt-2.2+C'-treated or 2000-rads-irradiated B6-pretreated B6D2F1 spleen cells. Both types of resistance are observed in B6D2F1 hybrids pretreated with more than 2.5 X 10(7) B6 spleen cells.     

Immunospecific depletion of graft-versus-host-reactive lymphocytes using sensitized syngeneic initiator T lymphocytes.

We investigated a model of a lethal graft-versus-host (GVH) reaction with the aim of depleting donor spleen cells of immunospecific GVH-reactive lymphocytes. In previous studies of the recruitment of effector T lymphocytes by sensitized syngeneic initiator T lymphocytes (ITLs) we found, using a local GVH reaction, that precursors of specific GVH-reactive lymphocytes were recruited to a draining lymph node. In this study, adult F1 hybrid mice were lethally irradiated and reconstituted with 2 x 10(6) syngeneic bone marrow cells and varying numbers of spleen cells from parental strain mice. To deplete donor spleen cells of GVH-reactive lymphocytes, parental strain mice were given injections in the hind footpads 6 days earlier of syngeneic ITLs that had been sensitized in vitro against allogeneic fibroblasts. We found that injection of ITLs sensitized against the relevant allogeneic antigens led to a marked decrease in the specific GVH potential of donor spleen cells. These findings show that GVH-reactive lymphocytes can be depleted selectively by activating their recruitment to particular lymph nodes, using syngeneic ITLs.     

Loss of F1 hybrid resistance to bone marrow grafts after injection of parental lymphocytes. Demonstration of parental anti-F1 T killer cells and general immunosuppression in the host

B6D2F1 mice acutely reject parental C57Bl/6 bone marrow grafts, a phenomenon that is known as hybrid resistance. Injection of C57Bl/6 splenocytes into B6D2F1 recipients prior to bone marrow transplantation had previously been shown to facilitate growth of C57Bl/6 marrow grafts. We show in this report that for this effect to occur, radiation-sensitive T cells have to be present in the splenocyte inoculum. Loss of hybrid resistance following injection of C57Bl/6 splenocytes into B6D2F1 mice coincides with the appearance of T killer cells of C57Bl/6 origin that are specific for H-2 histocompatibility antigens of DBA/2. The parental T killer cells in unresponsive B6D2F1 mice express in vitro cytotoxic activity on H-2d targets and appear to be responsible for the acquired in vivo rejection of H-2d bone marrow grafts. Appearance of donor-derived T killer cells coincides with marked suppression of host immunity: lymphocytes from unresponsive B6D2F1 mice do not proliferate in mixed lymphocyte reactions and fail to respond to sheep erythrocytes in vitro, nor do they express natural killer (NK) activity. Concomitant with the suppression of NK activity, hybrid resistance to C57B1/6 marrow grafts disappears. This loss of resistance to bone marrow transplants is unspecific since third-party SJL marrow grafts are not rejected. It is concluded that suppression of hybrid resistance by injection of parental splenocytes into B6D2F1 mice is caused by a severe nonspecific suppression of host immune responsiveness by parental T cells that recognize disparate histocompatibility antigens in the host.     

Alterations in T cell-derived colony-stimulating factors associated with GVH-induced immune deficiency

Injection of parental C57BL/10 spleen cells into unirradiated immune-competent (B10 x B10.BR)F1 hosts has been demonstrated to produce a graft-vs.-host-induced immune deficiency in T cell-mediated functions, including mitogen or alloantigen stimulated proliferation or cytotoxic T cell generation. The production of T cell-derived lymphokines affecting hematopoiesis was also altered during GVH. During the first two weeks of GVH, IL-3 and particularly GM-CSF were produced spontaneously; in subsequent weeks, the spontaneous production dropped to normal or subnormal levels. CSF content in concanavalin A-stimulated splenic supernatants was reduced at weeks 1-2, and declined to less than 5% of normal levels by 3-4 weeks of GVH. This decline in CSF content was correlated with a decrease in immune function as assessed by concanavalin A-stimulated IL-2 production and by generation of cytotoxic T lymphocytes. Concurrent with the recovery of immune function during GVH weeks 8-15, mitogen-stimulated production of CSF returned to normal levels. In addition to the decrease in CSF production identified in acute suppressive GVH, CSF content in concanavalin A-stimulated splenic supernatants was also decreased in chronic stimulatory GVH, generated in the strain combination (B6 x B6bm1)F1----(B6bm1 x B6bm12)F1. This decrease in CSF production correlated with a decrease in self-restricted T helper cell function. Finally, a decrease in both immune function and CSF production capacity was observed in the acute GVH following allogeneic (minor histocompatibility loci) bone marrow transplantation into irradiated hosts.     

Cytotoxic macrophage in graft-versus-host reaction.

Peritoneal macrophages of F1 hybrid mice injected with parental strain spleen cells 8-14 days previously ("early" graft-versus-host (GVH) macrophages) develop an increased ability to destroy in vitro bystanding target cells of syngeneic, allogeneic, and xenogeneic origin. At later stages of GVH reaction, the nonspecific cytotoxicity of macrophages wanes ("late" GVH macrophages) and does not differ much from that of control macrophages. Trypsinization of early GVH macrophages or iodoacetate treatment significantly reduces their cytotoxicity, whereas anti-theta serum and complement have only a moderate effect. Antimouse IgM antibody significantly enhanced the cytotoxic potential of early GVH macrophages, but had no effect either on normal or late GVH macrophages. In contrast, antimouse IgG antibody enhanced, although slightly, only the cytotoxic activity of late GVH macrophages. Two possible explanations of the increased cytotoxicity of early GVH macrophages are offered: (1) cytophilic antibody of IgM type, directed against host antigens, renders macrophages capable of damaging nonspecifically bystanding target cells upon contact with antigen; (2) IgM alloantibody produced by parental cells changes the surface properties of macrophages and contributes to the cytotoxicity of these cells.     

Lethal graft-versus-host reaction against non-H-2 antigens. I. Prevention of GVH-associated immunodeficiency by preimmunizing the donor against host-specific non-H-2 antigens

Injection of B10.D2 cells into irradiated H-2d compatible (DBA/2xB10.D2)F1 recipients provokes a lethal GVH that can be abrogated by donor preimmunization against host-specific DBA/2 non-H-2 antigens. To study the possible relationship between the observed protection and restoration of immune responsiveness, we compared spleen cellularity, selected T and B cell functions, and NK activity in GVH and protected mice during the 1st month after grafting. Normal and isografted mice served as controls. GVH was found to be characterized by an early stimulation phase associated with splenomegaly and increased percentages (but not numbers) of Lyt-2+ and L3T4+ cells, followed by profound aplasia and abrogation of IL-2 production. Response to a B cell mitogen (LPS) is depressed, and cells from GVH mice exert a strong suppressive effect on the LPS and PHA responsiveness of normal cells. Suppression appears to be mediated by a radioresistant, nylon nonadherent, asialo GM1 negative cell expressing a low level of Thy-1 antigen. In contrast, protection correlates with progressive restoration of spleen cellularity and LPS responsiveness, with decreased but clearly detectable IL-2 production, and transient nonspecific suppressor activity. The immune status of protected mice resembles that of isografted controls. No correlation was found between mortality (or protection) and either PHA responsiveness, which remained depressed in all grafted mice throughout the observation period, or NK activity, which was strongly depressed in both GVH and protected mice. In conclusion, protection correlates with the disappearance of nonspecific suppressor cells and the restoration of cellularity and certain nonspecific immune functions. Donor immunization against host-specific non-H-2 antigens, which protects against mortality, also protects against GVH-associated immune deficiency.     

Suppression of B lymphocyte genesis in the bone marrow by systemic graft-versus-host reactions

The effects of systemic graft-versus-host (GVH) reactions on B lymphocyte production in the bone marrow of mice were examined by quantitating populations of pre-B cells and B lymphocytes. Acute and chronic GVH reactions were induced by injecting A strain lymphoid cells into either (C57BL/6 X A) F1 or (CBA X A) F1 mice, respectively. Control groups of F1 hybrid mice were given syngeneic lymphoid cells. By double immunofluorescence labeling for cytoplasmic mu heavy chains of IgM (c mu) and for surface mu (s mu) the absolute numbers of pre-B cells (c mu + s mu-) and B lymphocytes (s mu +) in the bone marrow and spleen were determined. During acute GVH reactions, the pre-B cells and B lymphocytes in the bone marrow fell rapidly in numbers and were almost absent from 16 days until the end of the 30-day experimental period. In the spleen, the number of B lymphocytes remained normal for 8 days, then fell to less than 2% of control values from 16 days onward. A similar initial decline in pre-B cells and B lymphocytes occurred during chronic GVH reactions. In long-term survivors of GVH reactions, pre-B cells and B lymphocytes began to reappear after 40 days and maintained normal numbers from 100 to 150 days. The antibody response of spleen cells to sheep red blood cells was lost during GVH reactions. However, this occurred even before B lymphocytes were eliminated and the response remained subnormal after B lymphocyte numbers had recovered. The results demonstrate that systemic GVH reactions markedly depress the normally active genesis of primary B lymphocytes in the bone marrow of the host, accounting in part for the associated state of humoral immunodeficiency.     

CD28 knockout mice as a useful clue to examine the pathogenesis of chronic graft-versus-host reaction

BACKGROUND: Injection of BALB/c or DBA/2 spleen cells into F1 C57BL/6 (B6) hybrids induces a graft-versus-host reaction (GVHR) of a chronic stimulatory type that results in clinical and pathologic manifestations that resemble the human systemic lupus erythematosus (SLE). The aim of the present study was to examine the role of a major T-cell costimulatory signal receptor, CD28, in the production of autoantibody and the development of an immune complex glomerulonephritis, which are common in SLE pathology. METHODS: For this purpose, CD28-deficient (CD28KO) mice were used for the source of donor lymphocytes. Chronic GVHR was induced by an injection of BALB/c or BALB. CD28KO donor cells into normal BCF1 mice. Serum titers of anti-dsDNA antibodies were assessed by enzyme-linked immunosorbent assay (ELISA) and major histocompatibility complex (MHC) class II antigen expression on B cells were tested by flow cytometry. In addition, depositions of immunoglobulin (Ig) were examined by direct immunofluorescence staining on frozen kidney sections. RESULTS: When (BALB/c x B6)F1 mice were injected with parental BALB/c lymphocytes, serum anti-dsDNA titer was significantly increased in association with nonspecific B-cell activation and IgG deposition in the glomerular basement membrane. In sharp contrast, none of these signs were observed in F1 mice, which were injected with CD28KO spleen cells. CONCLUSION: The CD28-mediated T-cell costimulatory pathway plays a pivotal role in the development of polyclonal B-cell activation, autoantibody production, and an immune complex glomerulonephritis. We propose that CD28KO mice are useful clues in examining the pathogenesis of experimental lupus nephritis.     

Avoidance of graft versus host reactions in cured W-anemic mice.

Graft-versus-host reactions of parental cells in F1 hybrids were studied with two unrelated inbred strains of mice that differed at the mouse histocompatibility locus. W-anemic F1 recipients were compared with lethally irradiated normal F1 recipients. Both sets of recipients were populated by marrow and spleen cell grafts from parental and F1 donors. Most W-anemic F1 recipients were cured by parental and F1 cell grafts (except B6 spleen). Even after 13 to 18 months, they showed little or no effect from GVH reactions. Lethally irradiated normal F1 recipients tolerated parental marrow grafts almost as well, but gave dramatically different results with parental spleen grafts. Seventy-nine of 80 irradiated F1 recipients of parental spleen grafts died within 1 month. Unlike lethally irradiated recipients, W-anemic recipients have substantial numbers of their own cells along with the donor cells in their lymphoid tissues. These F1 lymphocytes may interact with parental lymphocytes in vivo to restrain reactions against F1 allogeneic antigens.     

Chimerism and cytotoxic T lymphocyte unresponsiveness after neonatal injection of spleen cells in mice. Effects of T cell depletion and of a semiallogeneic or fully allogeneic inoculum

Newborn Balb/c mice received a single neonatal injection of either (A/J x Balb/c) F1 hybrid spleen cells, T-cell-depleted (A/J x Balb/c) F1 hybrid spleen cells, or T-cell-depleted fully allogeneic A/J spleen cells. Chimerism was followed longitudinally during the life span by the detection of circulating donor allotype. At sacrifice, the percentage of donor cells in the spleen was measured, and cytotoxic T lymphocyte (CTL) reactivity to the tolerogen was tested. We found that T cell depletion of the semiallogeneic inoculum did not modify its capacity to generate persistent chimerism and CTL tolerance, while T-cell-depleted allogeneic cells were intrinsically deficient both in the induction and in the long-term maintenance of chimerism and CTL unresponsiveness.     

Hybrid resistance to parental bone marrow grafts in nonlethally irradiated mice

Resistance to parental bone marrow (BM) grafts in F1 hybrid recipients is due to natural killer (NK) cell–mediated rejection triggered through “missing self” recognition. “Hybrid resistance” has usually been investigated in lethally irradiated F1 recipients in conjunction with pharmacological activation of NK cells. Here, we investigated BM‐directed NK‐cell alloreactivity in settings of reduced conditioning. Nonlethally irradiated (1‐3 Gy) or nonirradiated F1 (C57BL6 × BALB/c) recipient mice received titrated doses (5‐20 x 10⁶) of unseparated parental BALB/c BM without pharmacological NK cell activation. BM successfully engrafted in all mice and multilineage donor chimerism persisted long‐term (24 weeks), even in the absence of irradiation. Chimerism was associated with the rearrangement of the NK‐cell receptor repertoire suggestive of reduced reactivity to BALB/c. Chimerism levels were lower after transplantation with parental BALB/c than with syngeneic F1 BM, indicating partial NK‐mediated rejection of parental BM. Activation of NK cells with polyinosinic–polycytidylic acid sodium salt poly(I:C), reduced parental chimerism in nonirradiated BM recipients but did not prevent hematopoietic stem cell engraftment. In contrast, equal numbers of parental lymph node cells were completely rejected. Hence, hybrid resistance leads to incomplete rejection of parental BM under reduced conditioning settings.