APCs in the liver and spleen recruit activated allogeneic CD8+ T cells to elicit hepatic graft-versus-host disease

Host APCs are required for initiating T cell-dependent acute graft-vs-host disease (GVHD), but the role of APCs in the effector phase of acute GVHD is not known. To measure the effect of tissue-resident APCs on the local development of acute GVHD, we selectively depleted host macrophages and DCs from the livers and spleens, but not from the skin, peripheral lymph nodes (PLN), or mesenteric lymph nodes (MLN), of C57BL/6 (B6) mice by i.v. administration of liposomal clodronate before allogeneic bone marrow transplantation. Depletion of host hepatic and splenic macrophages and DCs significantly inhibited the proliferation of donor C3H.SW CD8(+) T cells in the spleen, but not in the PLN or MLN, of B6 mice. Such organ-selective depletion of host tissue APCs also markedly reduced the trafficking of allogeneic CD8(+) T cells into the livers and spleens, but not PLN and MLN, of B6 recipients compared with that of the control mice. Acute hepatic, but not cutaneous, GVHD was inhibited as well, resulting in improved survival of liposomal clodronate-treated B6 recipients. When C3H.SW CD8(+) T cells were activated in normal B6 recipients, recovered, and adoptively transferred into secondary B6 recipients, activated donor CD8(+) T cells rapidly migrated into the livers and spleens of control B6 recipients but were markedly decreased in B6 mice that were depleted of hepatic and splenic macrophages and DCs. Thus, tissue-resident APCs control the local recruitment of allo-reactive donor T cells and the subsequent development of acute GVHD.     

Effect of graft-versus-host disease on anti-tumor immunity

BCL1, a spontaneous B cell leukemia of BALB/c origin, is rejected by C.B-20 (Ighb, H-40b) but not BALB/c (Igha, H-40a) mice. Adoptive transfer of C.B-20 anti-BCL1 effector cells specific for the minor histocompatibility Ag H-40a protects irradiated C.B-20 but not BALB/c recipients. Because C.B-20 donor cells could potentially generate graft-vs-host disease (GVHD) in BALB/c recipients, we investigated the possibility that GVHD prevents the anti-tumor effect. GVHD was induced in (C.B-20 X B10.D2)F1 [H-2d, H-40b X H-2d,H-40b] recipients after injection of B10.D2-primed C.B-20 donor cells. GVHD was indicated by the histologic appearance of tissue sections from C.B-20----F1 livers, target organs of GVHD, which showed a marked mononuclear cell infiltrate around the portal tracts and central veins. In addition, splenic lymphocytes from these mice had altered CD4/CD8 ratios and were unable to respond to the polyclonal activators Con A and LPS. The mitogen unresponsiveness was at least partially due to the presence of a suppressor cell, because proliferation of normal spleen cells to Con A and LPS was suppressed upon addition of C.B-20----F1 spleen cells. Further immune dysfunction was evident by the inability of T cells from mice with GVHD to generate a CTL response to H-2 alloantigens. Addition of C.B-20----F1 spleen cells to F1 responder cells at the induction of culture did not prevent generation of CTL, indicating that a suppressor cell was not responsible for the lack of CTL activity. In this setting of GVHD, F1 recipients were able to reject BCL1 upon adoptive transfer of C.B-20 anti-BCL1 effector cells. These data indicate that GVHD-induced immune dysfunction does not inhibit the activity of antileukemia T cells.     

Lethal graft-vs-host disease induced by a class II MHC antigen only disparity is not mediated by cytotoxic T cells

Treatment of C57BL/6J (B6) murine splenocytes with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) selectively removes NK cells, CTL precursors, and the capacity to cause lethal graft-vs-host disease (GVHD) in irradiated B6 X DBA/2 F1 mice. In contrast, alloantigen-induced L3T4(+) Th cell function has been shown to be relatively preserved after exposure to this agent. The present studies assessed the effects of Leu-Leu-OMe treatment of donor cells on induction of lethal GVHD in other murine strain combinations. When irradiated B6 X CBAF1 mice were infused with T and NK cell-depleted B6 bone marrow cells and 3 to 30 X 10(6) B6 spleen cells, uniformly lethal GVHD was observed. However, B6 X CBAF1 recipients of T and NK-depleted B6 bone marrow cells and similar numbers of Leu-Leu-OMe-treated B6 spleen cells demonstrated 90 to 100% long term survival. In contrast, Leu-Leu-OMe treatment of B6 donor cells had no beneficial effect on mortality rates in irradiated (B6 X B6-C-H-2bm12)F1 (B6 X bm12F1) recipients. When B6 spleen cells were stimulated in vivo or in vitro with either B6 X CBAF1 or B6 X bm12F1 stimulator cells, the capacity to generate alloantigen-specific CTL was abolished comparably by Leu-Leu-OMe treatment. Thus, the dramatic difference between the effects of Leu-Leu-OMe treatment of B6 spleen cells on the course of GVHD in B6 x CBAF1 and class II MHC only disparate B6 x bm12F1 recipients could not be explained by unique resistance of bm12-specific CTL precursors to Leu-Leu-OMe. These findings indicate that T cell effector mechanisms distinct from classic cell-mediated cytotoxicity are sufficient to generate lethal GVHD in class II MHC only disparate B6----B6 X bm12F1 mice.     

Mlsa generated suppressor cells. I. Suppression is mediated by double-negative (CD3+CD5+CD4-CD8-) alpha/beta T cell receptor-bearing cells.

Grafting of cells from B10.D2 (H-2d) donors into H-2 compatible lethally irradiated (DBA/2 x B10.D2)F1 hosts results in a severe graft-vs-host reaction (GVHR), developed against DBA/2 non-H-2 Ag, with only 0 to 10% of animals surviving. This GVHR mortality rate is dramatically reduced (90 to 100% of animals survive) by donor preimmunization against Mlsa determinants. The protection against GVHR correlates with a decreased B10.D2 anti-DBA/2 proliferative response in vitro. Both in vivo and in vitro phenomena are associated with activation of CD5+ suppressor T cells in the spleens of immunized mice. The present work was designed to study the origin of these suppressor cells and to further characterize their phenotype. The results show that significant suppression is not inducible in "B" mice. In contrast, in mice that were only thymectomized or else pretreated in vivo with anti-CD4 or anti-CD8 mAb, the suppressor cells are activated as efficiently as in normal mice. The suppression of GVHR mortality and proliferative responses in vitro is lost after depletion from preimmunized splenocytes of CD5+ T cells and remains unaltered after depletion of CD4+ or CD8+ T cells or both. Depletion of asialo GM1+ cells removes all NK activity, whereas the suppression is decreased only slightly. FACS analysis showed that double-negative (DN) cells from normal and immunized mice contain both CD3+ and CD3- cells; the vast majority of the CD3+ DN T cells express the alpha/beta T cell receptor. Suppression of GVHR and of proliferative responses in vitro are abrogated after elimination of CD3+ cells. These results suggest that Mlsa generated suppressor cells: 1) are derived from post-thymic long-lived T cell precursors; 2) are low asialo GM-1+ but do not exhibit NK activity; 3) belong to a subset of peripheral CD5+ DN T cells bearing a CD3-associated alpha/beta-heterodimer.     

G-CSF-treated donor CD4(+) T cells attenuate acute GVHD through a reduction in Th17 cell differentiation

The immunoregulatory effects of granulocyte colony-stimulating factor (G-CSF) on allogeneic peripheral blood cell transplantation (PBCT) have been demonstrated to reduce acute graft-versus-host disease (GVHD). However, the underlying mechanism is still not clear. In this study, we focused on the direct effects of G-CSF on donor CD4(+) T cell responses after transplantation. We observed that lethally irradiated B6D2F1 recipient mice that are transplanted with CD4(+) T cells from G-CSF-treated B6 donors showed mild attenuations in severity and mortality compared with recipients transplanted with PBS-treated CD4(+) T cells. Notably, skin GVHD was significantly reduced, but no such reduction was observed in other organs. Although there was no difference with respect to alloreactive expansion or Foxp3(+) Treg induction, the use of G-CSF-treated CD4(+) T cells significantly reduced the numbers of IL-17-producing and RORγt-expressing cells in the secondary lymphoid organs of allogeneic recipients after transplantation compared with the use of the control cells. Finally, we found that the suppressor of cytokine signaling-3 (SOCS3) expression in G-CSF-treated donor CD4(+) T cells was much higher than that in control CD4(+) T cells. Our results demonstrate that the inhibition of Th17 cell differentiation by SOCS3 induction is associated with the immunoregulatory role of G-CSF in CD4(+) T cell-mediated acute GVHD.     

Role of lymphokine-activated killer cells as mediators of veto and natural suppression

Fresh bone marrow cells have veto activity but little if any NK activity. By contrast, lymphokine-activated bone marrow cells have potent natural suppressor as well as veto activity, and also have cytolytic activity characteristic of lymphokine-activated killer cells. Veto activity of fresh bone marrow cells is eliminated by 9 Gy irradiation and by depletion of cells expressing Qa-2, but is unaffected by removal of cells expressing Thy-1, Qa-5, Ly-5, or asialo GM1. By contrast, the veto and NS activities of lymphokine-activated bone marrow cells are both abrogated by C lysis depletion of cells expressing Qa-2, Qa-5, Thy-1, asialo GM1, NK1, and Ly-11, but are unaffected by depletion of cells expressing Ly-2. Bone marrow cells depleted of Qa2+ cells fail to generate veto or natural suppressor activity when cultured in Con A-conditioned medium, unlike bone marrow cells depleted of mature NK1.1+ NK cells. Cloned NK cell line F8 is able to mediate both natural suppression and veto. These findings indicate that bone marrow veto and natural suppression are not mediated by T or NK cells present de novo in the bone marrow, but are dependent on proliferating cells that phenotypically resemble pre-NK cells. The progeny of these cells have the phenotype and functional activity of lymphokine-activated killer cells, and are capable of directly mediating both veto and natural suppression.     

Role of the passive apoptotic pathway in graft-versus-host disease

Donor T cells have been shown to undergo apoptosis during graft-vs-host disease (GVHD). Although active apoptosis mediated through Fas/Fas ligand interactions has been implicated in GVHD, little is known about the role of the passive apoptotic pathway. To examine this question, we compared the ability of normal donor T cells and T cells overexpressing the antiapoptotic protein, Bcl-x(L), to mediate alloreactive responses in vitro and lethal GVHD in vivo. In standard MLCs, T cells that overexpressed Bcl-x(L) had significantly higher proliferative responses but no difference in cytokine phenotype. Overexpression of Bcl-x(L) prolonged survival of both resting and alloactivated CD4(+) and CD8(+) T cells as assessed by quantitative flow cytometry, accounting for the higher proliferative responses. Analysis of engraftment in murine transplantation experiments demonstrated an increase in donor T cell chimerism in animals transplanted with Bcl-x(L) T cells, suggesting that overexpression of Bcl-x(L) prolonged T cell survival in vivo as well. Notably, transplantation of Bcl-x(L) T cells into nonirradiated F(1) recipients also significantly exacerbated GVHD as assessed by mortality and pathological damage in the gastrointestinal tract. However, when mice were irradiated no difference in GVHD mortality was observed between animals transplanted with wild-type and Bcl-x(L) T cells. These data demonstrate that the passive apoptotic pathway plays a role in the homeostatic survival of transplanted donor T cells. Moreover, the susceptibility of donor T cells to undergo passive apoptosis is a significant factor in determining GVHD severity under noninflammatory but not inflammatory conditions.     

Donor CD8+ T cells mediate graft-versus-leukemia activity without clinical signs of graft-versus-host disease in recipients conditioned with anti-CD3 monoclonal antibody

Donor CD8(+) T cells play a critical role in mediating graft-vs-leukemia (GVL) activity, but also induce graft-vs-host disease (GVHD) in recipients conditioned with total body irradiation (TBI). In this study, we report that injections of donor C57BL/6 (H-2(b)) or FVB/N (H-2(q)) CD8(+) T with bone marrow cells induced chimerism and eliminated BCL1 leukemia/lymphoma cells without clinical signs of GVHD in anti-CD3-conditioned BALB/c (H-2(d)) recipients, but induced lethal GVHD in TBI-conditioned recipients. Using in vivo and ex vivo bioluminescent imaging, we observed that donor CD8(+) T cells expanded rapidly and infiltrated GVHD target tissues in TBI-conditioned recipients, but donor CD8(+) T cell expansion in anti-CD3-conditioned recipients was confined to lymphohematological tissues. This confinement was associated with lack of up-regulated expression of alpha(4)beta(7) integrin and chemokine receptors (i.e., CXCR3) on donor CD8(+) T cells. In addition, donor CD8(+) T cells in anti-CD3-conditioned recipients were rendered unresponsive, anergic, Foxp3(+), or type II cytotoxic T phenotype. Those donor CD8(+) T cells showed strong suppressive activity in vitro and mediated GVL activity without clinical signs of GVHD in TBI-conditioned secondary recipients. These results indicate that anti-CD3 conditioning separates GVL activity from GVHD via confining donor CD8(+) T cell expansion to host lymphohemological tissues as well as tolerizing them in the host.     

TNF enhances CD4+ T cell alloproliferation,IFN-gamma responses,and intestinal graft-versus-host disease by IL-12-independent mechanisms

Inhibition of TNF/TNFR2 interactions ameliorates intestinal graft-vs-host disease (GVHD) and Th1 cytokine responses induced by transfer of B6 CD4(+) spleen cells into irradiated MHC class II disparate B6.C-H-2(bm12) (bm12) x B6 F(1) recipients. The present studies examined whether these effects of TNF are IL-12 dependent. T cell proliferative responses of B6.129S1-IL-12rb2(tm1Jm) (B6.IL-12R(-/-)) responder spleen cells were found to be comparable to those of control B6 spleen cells. TNF inhibition reduced T cell proliferation and IFN-gamma production in supernatants of MLC using either B6.IL-12R(-/-) or control B6 responder cells. GVHD induced wasting disease in recipients of B6.IL-12R(-/-) CD4(+) spleen cells that received a TNF inhibitor-encoding adenovirus (5.4 +/- 6.5% weight loss (n = 7)) was significantly reduced compared with levels of weight loss observed in recipients that had received a control adenovirus (25.7 +/- 12.2% weight loss (n = 11), p = 0.001). Furthermore, TNF inhibition was associated with a reduction in colonic GVHD scores (p = 0.039) and in the percentage of the splenic CD4(+) T cells that expressed IFN-gamma (16 vs 6%). These findings indicate that TNF promotes CD4(+) T cell alloproliferation, IFN-gamma responses, and intestinal GVHD by IL-12-independent mechanisms.     

Effector and regulatory cells in autoimmune oophoritis elicited by neonatal thymectomy.

(C57BL/6 x A/J)F1 (B6AF1) mice thymectomized between days 1 and 4 of age develop autoimmune oophoritis (D3TX oophoritis) 4 to 6 wk later. Oophoritis can be adoptively transferred to young recipients, and the disease in D3TX mice is prevented by reconstitution with normal adult spleen cells. The present study was further defined the nature of the effector and suppressor cells. Contrary to an earlier report, oophoritis is transferred to syngeneic and not allogeneic recipients. The spleen cells from D3TX mice when stimulated in vitro with Con A, also transfer oophoritis to adult recipients. The effector cells are CD4+: oophoritis transfer is abrogated by CD4 antibody and not by CD8 antibody and C. Spleen cells from D3TX male mice transfer disease less efficiently than female cells, thus endogenous ovarian Ag may be required for activation of effector T cells. T cells from normal adult spleen that suppress D3TX oophoritis also appear to be of CD4+ phenotype. These cells are likely to be derived from adult thymus because adult thymocytes also suppress D3TX disease. We were unable to substantiate the earlier claim that suppressor cells in normal mice are ovarian Ag specific. Thus male and female spleen cells suppress disease with comparable efficiency, and deprivation of endogenous ovarian Ag by neonatal ovariectomy of cell donors had no observable effect on disease suppression.     

Opposing effects of ICOS on graft-versus-host disease mediated by CD4 and CD8 T cells

ICOS, a CD28 family member expressed on activated CD4(+) and CD8(+) T cells, plays important roles in T cell activation and effector function. Here we studied the role of ICOS in graft-vs-host disease (GVHD) mediated by CD4(+) or CD8(+) T cells in allogeneic bone marrow transplantation. In comparison of wild-type and ICOS-deficient T cells, we found that recipients of ICOS(-/-) CD4(+) T cells exhibited significantly less GVHD morbidity and delayed mortality. ICOS(-/-) CD4(+) T cells had no defect in expansion, but expressed significantly less Fas ligand and produced significantly lower levels of IFN-gamma and TNF-alpha. Thus, ICOS(-/-) CD4(+) T cells were impaired in effector functions that lead to GVHD. In contrast, recipients of ICOS(-/-) CD8(+) T cells exhibited significantly enhanced GVHD morbidity and accelerated mortality. In the absence of ICOS signaling, either using ICOS-deficient donors or ICOS ligand-deficient recipients, the levels of expansion and Tc1 cytokine production of CD8(+) T cells were significantly increased. The level of expansion was inversely correlated with the level of apoptosis, suggesting that increased ability of ICOS(-/-) CD8(+) T cells to induce GVHD resulted from the enhanced survival and expansion of those cells. Our findings indicate that ICOS has paradoxical effects on the regulation of alloreactive CD4(+) and CD8(+) T cells in GVHD.     

CD11 molecule defines two types of suppressor cells within the T8+ population

It has been shown that T8+ cells are comprised of functionally heterogeneous subpopulations such as suppressor, cytotoxic, and NK cells. In this report, we attempted to delineate the functional heterogeneity of T8 cells defined by anti-CD11 antibody (anti-Mol). Although allospecific cytotoxic activity was restricted to the T8+Mol- subset, suppression of PWM IgG synthesis could be elicited in both the T8+Mol+ and the T8+Mol- subset of cells. However, the mechanism of suppression was different in these two subsets. Suppression by the T8+Mol- subset of cells required the interaction with the T4+2H4+ suppressor inducer cells, whereas the T8+Mol+ subset of cells could suppress in the absence of the suppressor inducer cells. Moreover, recombinant interleukin-2 alone could augment this suppression by the T8+Mol+ subset, but did not induce suppression by the T8+Mol- subset. In contrast, NK and LAK activity was exclusively found in the T8+Mol+ subset of cells but not in the T8+Mol- subset of cells. These results suggest that the CD11 molecule is useful for distinguishing novel subsets of T8 cells.     

In vitro characterization of a murine recipient anti-donor effector cell responsible for the development of chronic graft-versus-host disease

Injection of parental spleen cells into unirradiated F1 hybrid recipients is frequently used for the induction of murine graft-vs-host disease (GVHD). Injection of C57BL/6 spleen cells into B6D2F1 recipients results in the acute form of GVHD whereas injection of DBA/2J cells into the same recipients results in a chronic form of GVHD. In vivo studies have suggested that injection of DBA/2J cells into B6D2F1 recipients results in the chronic form of GVHD instead of acute GVHD because of recipient B6D2F1 effector cells that inhibit DBA/2J CD8+ T cell function. The present report describes the development of an in vitro assay that measures the activity of these B6D2F1 effector cells. The results obtained using this assay indicate that these B6D2F1 effector cells are found primarily in the LN, need to be activated in order to function, are inhibited by anti-CD8 antibodies, and mediate their function as a result of DBA/2J cells binding to Ag expressed on their cell surface.     

In vivo induced allo-reactive natural killer cells.

We have previously shown that rat allo-selective cells of the CD2+CD5- phenotype were generated in Brown Norway (BN) rats after immunization with allogeneic Wistar/Furth (WF) cells, whereas immunization with semi-allogeneic F1 (WF/BN) cells generated CD2+CD5+ effector T cells. We now report that the allo-selective CD2+CD5- lymphocytes lacked expression of intact CD3 complexes and expressed NKR-P1 molecules although lower as compared to classical NK cells, implicating that these lymphocytes constitute a subset of NK cells. The CD5+ T cells were not cytolytically active in BN rats immunized with WF cells indicating an intersubset regulation with mutually exclusive activation of either allo-selective T cells or allo-selective NK cells. Cold target inhibition showed that lysis of both allogeneic target cells and NK-sensitive target cells was mediated by the same NKR-P1 intermediate effector cells. These NK cells lysed WF but not allogeneic Fischer 344 or autologous BN target cells, indicating selective recognition of an allogeneic determinant. Semiallogeneic F1 (WF/BN) target cells were not lysed. Furthermore, target cells from F1 (WF/BN) x WF back-cross hybrids lacking expression of RT1n (self-MHC class I) were susceptible to lysis, whereas back-cross hybrids expressing RT1n were protected from lysis, indicating that self-MHC molecules conferred protection from lysis. These findings implicate the existence of NKR-P1intermediate and NKR-P1high NK cell subsets with different regulation and function in vivo.     

CD103 deficiency prevents graft-versus-host disease but spares graft-versus-tumor effects mediated by alloreactive CD8 T cells

Background

Graft-versus-host disease (GVHD) remains the main barrier to broader application of allogeneic hematopoietic stem cell transplantation (alloSCT) as a curative therapy for host malignancy. GVHD is mediated by allogeneic T cells directed against histocompatibility antigens expressed by host tissues. Based on previous studies, we postulated that the integrin CD103 is required for CD8-mediated GVHD, but not for graft-versus-tumor effects (GVT).

Methodology/Principal Findings

We herein provide evidence in support of this hypothesis. To circumvent the potentially confounding influence of donor CD4 T cells, we developed an alloSCT model in which GVHD mortality is mediated by purified CD8 T cells. In this model, host-reactive CD8 T cells receive CD4 T cell help at the time of initial activation but not in the effector phase in which mature CD8 T effectors migrate into host tissues. We show that donor CD8 T cells from wild-type BALB/c mice primed to host alloantigens induce GVHD pathology and eliminate tumors of host origin in the absence of host CD4 T cells. Importantly, CD103 deficiency dramatically attenuated GVHD mortality, but had no detectable impact on the capacity to eliminate a tumor line of host origin. We provide evidence that CD103 is required for accumulation of donor CD8 T cells in the host intestinal epithelium but not in the tumor or host lymphoid compartments. Consistent with these data, CD103 was preferentially expressed by CD8 T cells infiltrating the host intestinal epithelium but not by those infiltrating the tumor, lamina propria, or lymphoid compartments. We further demonstrate that CD103 expression is not required for classic CD8 effector activities including cytokine production and cytotoxicity.

Conclusions/Significance

These data indicate that CD103 deficiency inhibits GVHD pathology while sparing anti-tumor effects mediated by CD8 T cells, identifying CD103 blockade as an improved strategy for GVHD prophylaxis.     

A SCID patient reconstituted with HLA-incompatible fetal stem cells as a model for studying transplantation tolerance.

We studied a severe combined immunodeficiency (SCID) patient who received transplantations with completely HLA-mismatched fetal liver and thymus from two different donors. The patient is now 14 years old, healthy and shows normal immunoresponses to recall antigens. His T cells are of donor origin, whereas the monocytes, B cells, and natural killer (NK) cells are of the recipient. The successful immunological reconstitution raised questions as to how T and B cells could collaborate across an HLA barrier and how tolerance was achieved. We have shown that tetanus toxin-specific T cell clones isolated from this patient recognized this antigen in the context of host and not of donor HLA-DR, indicating that those cells were educated in the host environment, presumably the thymus. Despite this, an unexpectedly high frequency of host-reactive clones was found that could recognize MHC antigens of the host. It was particularly striking that CD8+ CTL clones were obtained that recognized class I MHC antigens on the host cells. Nevertheless, the patient did not show any sign of acute or chronic graft-versus-host disease (GVHD). These data indicated that no or only incomplete clonal deletion had taken place in this patient and suggest the presence of a peripheral suppressor mechanism. Thus far, we have no indication for the existence of suppressor T cells. Inasmuch as it was found that host-reactive T cells fail to produce IL-4, which is exceptional for CD4+ T cells, we are exploring the possibility that abnormal cytokine production patterns of host-reactive T cells are associated with suppression of these cells in vivo.     

Immunodeficiency in graft-versus-host disease. I. Mechanism of immune suppression

Irradiated CBA/J mice transplanted with H-2 compatible, minor histocompatibility disparate B10.BR bone marrow develop graft-versus-host disease (GVHD) if mature T lymphocytes are added to the marrow inoculum. In the setting of mild GVHD (receiving 10(4) or 10(5) T cells), by phenotypic analysis, lymphoid reconstitution occurs normally within 4 to 6 wk but there is a profound deficiency in the ability of splenic lymphocytes to respond to polyclonal activators such as LPS and Con A. This unresponsiveness is attributable to active suppression mediated by cells that express Thy-1 and can be removed with leucine methyl ester treatment. Thus, splenocytes from mice with GVHD suppress responses of normal T and B lymphocytes. Moreover, depletion of these suppressor cells restores normal function to splenocytes from mice with GVHD, and B cells isolated from these mice respond normally to T-dependent and -independent stimulation. Finally, IFN-gamma plays an important role in this suppression, because a neutralizing anti-IFN-gamma mAb significantly removes suppression of normal cells and restores functional responses of lymphocytes from mice with GVHD. These results provide insights into the mechanisms of immunodeficiency associated with GVHD, and suggest novel strategies for possible therapies for this disorder.     

B-cell-mediated regulation of delayed-type hypersensitivity

We obtained immune sera from mice which received suppressor B cells induced in vitro, injected them into immunized mice, and measured suppression of the delayed-type hypersensitivity (DTH) of these recipient mice. In the recipients, effector-phase suppressor T (Ts) cells were induced, and the action of these Ts cells was antigen-nonspecific. The suppressive material of the sera was adsorbed on a Sepharose column coated with anti-mouse immunoglobulin antibody and acid elution of the column yielded the elute fraction that showed significant suppressive activity. The suppressive activity of the sera was also adsorbed by an antigen-coated Sepharose column, and the eluate from the column had suppressive activity. Moreover, we established antigen-specific monoclonal antibodies, some of which suppressed the DTH in an H-2-nonrestricted way. The isotype or specificity of the antibodies was not related to the suppression, because suppressive and nonsuppressive antibodies belonged to the same immunoglobulin isotype and because the antibodies that recognized the same epitope had different suppressive activities. The Fc portion was not the functional site, because the F(ab')2 fragment had the activity. The suppressive antibody induced effector-phase Ts cells, which had the anti-idiotypic receptor. These findings suggested that antigen-specific antibodies in the immune sera mediated the suppression of DTH by the induction of effector-phase Ts cells in vivo and the idiotype of the antibody stimulated the anti-idiotypic receptor of these Ts cells.     

Specific inhibition of hybrid resistance in F1 hybrid mice pretreated with parent-strain spleen cells. III. Study of the mechanism

The rejection of H-2b parental bone marrow graft by lethally irradiated F1 recipients, that is known as hybrid resistance (HR), is a multistep process. In a first step a 5-fluorouracil (5-FU)-sensitive T cell recognizes the parental bone marrow cells and stimulates a macrophage-like cell to secrete IFN-alpha/beta (recognition phase). IFN-alpha/beta in turn activates a cyclophosphamide-sensitive NK-like cell that is the effector cell for HR (effector phase). In a previous paper we described that HR is specifically abrogated by the pretreatment of the F1 recipient with H-2b parental spleen cells. This abrogation is due to a Thy-1+CD5+CD4+CD8- nylon adherent suppressor cell of F1 origin. The aim of the present work was to study during which of the different phases of HR the activity of the suppressor cell is exerted. Our results showed that abrogation of HR in (C57BL/6 x C3H)F1 (B6C3F1) hybrids pretreated with B6 spleen cells results from: 1) the suppression of the 5-FU-sensitive T cell; 2) the suppression of the cyclophosphamide-sensitive NK-like cell; and 3) the disappearance of a humoral factor that is present in the serum of normal B6C3F1 hybrids and which seems to be involved in the effector phase of HR. The 5-FU-sensitive T cell is the only target of Thy-1+CD5+CD4+CD8- suppressor cell. The mechanisms responsible for the suppression of the NK-like effector cell and the disappearance of the humoral factor are discussed.     

Role of immunoregulatory donor T cells in suppression of graft-versus-host disease following donor leukocyte infusion therapy

In murine models of allogeneic bone marrow transplantation (BMT), MHC-mismatched recipients given a delayed infusion of donor leukocytes (DLI) at 21 days posttransplant develop significant GVHD whereas MHC-matched recipients do not. The current study was initially designed to test the hypothesis that small numbers of T cells in the MHC-mismatched donor bone marrow (BM) graft exacerbated graft-vs-host disease (GVHD) when DLI was administered at 21 days after BMT. Ex vivo depletion of Thy1+ cells from the donor BM had no impact on the severity of GVHD after DLI. However, depletion of donor T cells in vivo with a Thy1 allele-specific mAb given after BMT resulted in significantly more severe GVHD after DLI. Similar results were obtained in a MHC-matched model of allogeneic BMT, indicating that this was a general phenomenon and not model dependent. These results indicated that a population of donor-derived Thy1+ cells suppressed graft-vs-host reactivity after DLI. Results of experiments with thymectomized recipients demonstrated that an intact thymus was required for generation of the immunoregulatory donor cells. Experiments using TCR beta-chain knockout mice as BM donors indicated that the immunosuppressive Thy1+ cells coexpressed alphabetaTCR heterodimers. Similar experiments with CD4 and CD8 knockout donor BM suggested that the immunoregulatory Thy1+alphabetaTCR+ cells consisted of two subpopulations: a CD4+CD8- subpopulation and a CD4-CD8- subpopulation. Together, these results show that thymus-derived, Thy1+alphabetaTCR+ donor cells generated early after allogeneic BMT suppress the graft-vs-host reactivity of T cells given as DLI. These cells may mediate dominant peripheral tolerance after allogeneic BMT.