Numerical impairment of nestin(+) bone marrow niches in acute GvHD after allogeneic hematopoietic stem cell transplantation for AML.

The nestin(+) perivascular bone marrow (BM) stem cell niche (N(+)SCN) may be involved in GvHD. To investigate whether acute GvHD (aGvHD) reduces the number of N(+)SCN, we examined patients with AML who had undergone allogeneic hematopoietic stem cell transplantation. In the test cohort (n=8), the number of N(+)SCN per mm(2) in BM biopsies was significantly reduced in aGvHD patients at the time of aGvHD compared with patients who did not have aGvHD (1.2±0.78 versus 2.6±0.93, P=0.04). In the validation cohort (n=40), the number of N(+)SCN was reduced (1.9±0.99 versus 2.6±0.90 N(+)SCN/mm(2), P=0.05) in aGvHD patients. Receiver operating curves suggested that the cutoff score that best discriminated between patients with and without aGvHD was 2.29 N(+)SCN/mm(2). Applying this cutoff score, 9/11 patients with clinically relevant aGvHD (⩾grade 2) and 13/20 with any type of GvHD had decreased N(+)SCN numbers compared with only 10/29 patients without clinically relevant aGvHD (P=0.007) and 6/20 patients without any type of GvHD (P=0.028). In patients tracked over time, N(+)SCN density returned to normal after aGvHD resolved or remained stable in patients who did not have aGvHD. Our results show a decrease in the number of N(+)SCN in aGvHD.

[Foreign and self--the challenge for the immune system]. / Fremd und Selbst--Die Herausforderung für das Immunsystem.

This process of thymic selection produces a repertoire of T-cells able to recognize foreign antigens but to remain nonreactive to the body's own antigens, designated "self" (central tolerance). The interaction between lymphoid and stromal cells within the thymic microenvironment is critical in order to shape the correct antigen specificity of the T-cell repertoire. The thymus has been regarded as the main site of tolerance induction to ubiquitous self-antigens, i.e. abundant blood-borne self-antigens or proteins that are expressed by thymic cells. In contrast, tolerance to ubiquitous, i.e. tissue-restricted, self-antigens has been attributed to extrathymic, peripheral mechanisms. Here I review scientific evidence which assigns a much larger role for the thymus in tissue-specific tolerance. The reason for this paradigmatic shift is, first, the discovery of ectopic expression of tissue-restricted self-antigens by thymic medullary epithelial cells (TECs) which thus present to developing T-cells a "molecular mirror of peripheral self". As a consequence, central tolerance to many peripheral self-antigens can be generated. Second, the thymus is capable to produce regulatory T-cells. Such CD4+CD25+Foxp3+ T-cells are selected by antigens presented by medullary TECs and can control in the periphery the activation of tissue-specific self-reactive T-cells by use of active inhibitory mechanisms. These new insights have implications for our understanding of self-tolerance and its breakdown in autoimmune diseases.

Thymic atrophy in murine acute graft-versus-host disease is effected by impaired cell cycle progression of host pro-T and pre-T cells.

Reconstitution of the peripheral T-cell compartment is a critical aspect for the success of bone marrow transplantation and is also dependent on the reestablishment of normal thymic structure and function. Graft-versus-host disease (GVHD), however, exacerbates posttransplant immunodeficiency through a deleterious effect on thymic function. To investigate the mechanisms of GVHD-mediated thymic disease, 2 murine parent-->F(1 )transplantation models of acute and chronic GVHD, respectively, were studied. Acute GVHD was associated with changes in thymic architecture and a reduction in cellularity mainly because of the decrease in CD4(+)CD8(+), or double-positive (DP) thymocytes, to less than 15% of values found in mice without GVHD. Simultaneously, mature donor-derived T cells expanded in the confines of the allogeneic thymic microenvironment, leading to local inflammation. Through analysis of in vivo cell proliferation, we demonstrated that the ensuing depletion of DP thymocytes was secondary to a decreased commitment of resident pro-T and pre-T cells to enter the cell cycle. Moreover, DP cells themselves showed altered proliferative capacities in the presence of acute GVHD. These findings suggested that thymic atrophy in acute GVHD is effected by impaired cellular proliferation of immature host thymocytes and that the failure of these cells to enter the cell cycle is dependent on an interferon (IFN)-gamma-driven immune response. In contrast, interleukin-4-driven chronic GVHD was not accompanied by a sustained thymic infiltration of donor T cells. Consequently, there was a lack of apparent structural changes, a restricted in situ transcription of inflammatory cytokines, and a virtually unchanged cell cycle progression in vivo.

Apoptosis of thymocytes during acute graft-versus-host disease is independent of glucocorticoids.

BACKGROUND: Elimination of immature thymocytes resulting in thymic atrophy is characteristic of acute graft-versus-host disease (aGVHD). Because aGVHD has been associated with elevated glucocorticoid (GC) production, and CD4,CD8 double-positive thymocytes undergo rapid apoptosis in response to GCs, we hypothesized that administration of the GC receptor antagonist RU486 (mifepristone) should alter aGVHD-mediated thymocyte apoptosis. METHODS: Thymic development in the presence of aGVHD was studied in a haploidentical nonirradiated murine transplantation model (C57BL/6-->B6D2F1). Recipients were treated with RU486 or vehicle alone. Thymic development was analyzed by flow cytometry at different times post transplant. RESULTS: Acute thymic GVHD was characterized (1) by infiltration of mature donor-derived T cells and (2) by increased apoptosis of immature CD4+CD8+ thymocytes between 1 and 2 weeks after allogeneic transplantation. Contrary to expectations, administration of RU486 had no effect on these two parameters. CONCLUSIONS: Our data suggest that thymic pathology during aGVHD is mediated via a glucocorticoid-independent mechanism of apoptosis as blockade of glucocorticoid receptors did not alter the GVHD-induced thymic phenotype.

Contribution of CD40-CD154-mediated costimulation to an alloresponse in vivo.

BACKGROUND: Costimulation through CD40-CD154 plays an important role in T-cell activation. Although systemic administration of anti-CD154 antibody prevents or delays rejection of organ allografts in animal models, the molecular mechanisms responsible for this effect are not well defined. METHODS: We have previously demonstrated that priming of mice (H2d) with CD40-/- but not with wildtype naive B cells (H2b) leads to alloantigen-specific T-cell hyporesponsiveness in vitro. In the present study, we investigated whether such priming modifies allograft rejection in a major histocompatibility complex-mismatched murine cardiac transplantation model. RESULTS: Priming of hosts with donor-specific CD40-/- B cells delayed rejection of subsequently transplanted wild-type cardiac allografts by 8.0 days (P<0.001). The lack of CD40 on the cardiac graft delayed rejection in unprimed or primed hosts by 3-5 days. Prolongation of graft survival correlated with the failure of infused CD40-/- B cells to express B7.2 and ICAM-1 in vivo. CONCLUSIONS: Our data suggest that CD40-CD154 costimulation contributes to T cell priming to alloantigens in vivo and to a second set rejection phase in which donor antigens are presented to primed T cells.

Host reactive donor T cells are associated with lung injury after experimental allogeneic bone marrow transplantation.

Noninfectious lung injury is common after allogeneic bone marrow transplantation (BMT), but its association with acute graft-versus-host disease (GVHD) is unclear. Using a murine BMT system where donor and host differ by multiple minor histocompatibility (H) antigens, we investigated the nature of lung injury and its relationship both to systemic GVHD and host-reactive donor T cells. Lethally irradiated CBA hosts received syngeneic BMT or allogeneic (B10.BR) T-cell-depleted (TCD) bone marrow (BM) with and without the addition of T cells. Six weeks after BMT, significant pulmonary histopathology was observed in animals receiving allogeneic BMT compared with syngeneic controls. Lung damage was greater in mice that received allogeneic T cells and developed GVHD, but it was also detectable after TCD BMT when signs of clinical and histologic acute GVHD were absent. In each setting, lung injury was associated with significant alterations in pulmonary function. Mature, donor (Vbeta6(+) and Vbeta3(+)) T cells were significantly increased in the broncho-alveolar lavage (BAL) fluid of all allogeneic BMT recipients compared with syngeneic controls, and these cells proliferated and produced interferon-gamma (IFN-gamma) to host antigens in vitro. These in vitro responses correlated with increased IFN-gamma and tumor necrosis factor-alpha (TNF-alpha) in the BAL fluid. We conclude that alloreactive donor lymphocytes are associated with lung injury in this allogeneic BMT model. The expansion of these cells in the BAL fluid and their ability to respond to host antigens even when systemic tolerance has been established (ie, the absence of clinical GVHD) suggest that the lung may serve as a sanctuary site for these host reactive donor T cells. These findings may have important implications with regard to the evaluation and treatment of pulmonary dysfunction after allogeneic BMT even when clinical GVHD is absent.

CD8+ T cells activated during the course of murine acute myelogenous leukemia elicit therapeutic responses to late B7 vaccines after cytoreductive treatment.

We have previously shown in a murine acute myelogenous leukemia (AML) model that leukemic mice can be cured with a B7 vaccine if immunized early in the disease and that CD8+ T cells are necessary for tumor rejection. However, when B7 vaccine is administered 2 weeks after leukemia inoculation, the effect is only prolonged survival, ending in death virtually of all the mice. To distinguish between tumor kinetics and tumor-induced immunosuppression as potential mechanisms eliminating the therapeutic potential of late B7 vaccines, we performed in vitro T-cell studies during leukemia progression and in vivo studies on the clinical outcome of late B7 vaccines in combination with prior cytoreductive chemotherapy. Our results show that CD8+ T cells from leukemic mice 1 and 2 weeks after leukemia inoculation proliferate more vigorously in response to in vitro activation than cells from normal mice and produce Th1-type cytokines interleukin-2 and interferon-gamma. Cytotoxic T lymphocyte (CTL) assays demonstrate that cells from week-2 vaccinated mice (which succumb to their leukemia), surprisingly develop a stronger CTL activity than cells from week-1 vaccinated mice (which reject their leukemia). Finally, the combination of late chemotherapy and late B7 vaccine administration can cure only 20% of leukemic mice, whereas early chemotherapy and the same late B7 vaccine administration cure 100% of leukemic mice. These results demonstrate that in murine AML tumor growth does not induce T-cell anergy or a Th2 cytokine profile and suggest that tumor growth is most likely to be the limiting factor in the curative potential of late B7 vaccines.

Cytokine Dysregulation in Acute Graft-versus-Host Disease.

Giaft versus host disease (GVHD) remains the principal complication limiting the wider application of allogeneic bone marrow transplantation (BMT). Advances in basic immunology during the last decade have demonstrated how interactions between immunologically competent cells are governed by cytokines, and much recent research has focused on the roles of these mediators in the pathogenesis of acute GVHD. This article will review current evidence that dysregulated cytokine production can be considered a cascade of sequential activation of T-cells and monocytes that is responsible for many of the manifestations of acute GVHD. We suggest that cytokine dysregulation can be divided into three phases. Phase 1 is initiated by the conditioning of the host, which induces inflammatory processes in recipient tissues. Donor T-cell activation by host alloantigens and subsequent cytokine secretion in Phase 2 is facilitated by the consequences of Phase 1. The T-cell derived cytokines of Phase 2 activate distal inflammatory meditators which, together with T and NK-cell-mediated cytotoxicity, produce the systemic morbidity of GVHD-associated immunosuppression in Phase 3. Data from both experimental and clinical studies involving cytokines and their blockade in the prevention or treatment of GVHD will be reviewed.

The role of cytokines in acute graft-versus-host disease.

Graft-versus-host disease (GVHD) remains the principal complication limiting the wider application of allogeneic bone marrow transplantation (BMT). Advances in basic immunology during the last decade have demonstrated how interactions between immunologically competent cells are governed by cytokines, and much recent research has focused on the roles of these mediators in the pathogenesis of acute GVHD. This article reviews current evidence that dysregulated cytokine production can be considered a cascade of sequential monocyte and T-cell activation that is responsible for many of the manifestations of acute GVHD. We suggest that cytokine dysregulation can be conceptualized in three phases. Phase 1 is initiated by the conditioning of the host, which induces inflammatory processes in recipient tissues. Donor T-cell activation by host alloantigens and subsequent cytokine secretion in phase 2 is facilitated by the consequences of phase 1. The T-cell-derived cytokines of phase 2 activate distal inflammatory mediators, which, in synergy with T- and NK-cell-mediated cytotoxicity, produce the systemic morbidity of GVHD-associated immunosuppression in phase 3. Data from both experimental and clinical studies involving cytokines and their blockade in the prevention or treatment of GVHD are reviewed.

Transplantation of polarized type 2 donor T cells reduces mortality caused by experimental graft-versus-host disease.

Acute graft-versus-host disease (GVHD) is thought to be initiated by alloreactive type 1 T cells that secrete gamma-interferon (IFN-gamma). IFN-gamma induces the production of inflammatory cytokines, e.g., tumor necrosis factor-alpha and interleukin (IL)-1, which are the distal mediators of GVHD. We demonstrate that the transplantation of polarized type 2 murine T cells (i.e., cells secreting IL-4 but not IFN-gamma) together with T-cell-depleted bone marrow results in a significant increase in survival (P<0.001) after bone marrow transplantation across minor histocompatibility barriers (B10.BR-->CBA/J). Further analysis demonstrated that increased survival in recipients of polarized type 2 T cells correlated with diminished production of both IFN-gamma and tumor necrosis factor-alpha but with increases in IL-4 2 weeks after transplantation. Despite improved survival, histologic changes of GVHD were evident in oral mucosal and hepatic tissues at 7 weeks after bone marrow transplantation. These data provide further evidence that inflammatory cytokines in the immediate posttransplant period are pivotal to the development of mortality but that they do not correlate with individual target organ damage.

Long-term engraftment, graft-vs.-host disease, and immunologic reconstitution after experimental transplantation of allogeneic peripheral blood cells from G-CSF-treated donors.

Peripheral blood cells (PBPC) are an alternative source of bone marrow for allogeneic transplantation. Reports from recent clinical trials granulocyte colony-stimulating factor (G-CSF)-mobilized PBPC for allogeneic transplantation show incidence and severity of graft-vs.-host disease (GVHD) similar to those observed in conventional bone marrow transplantation (BMT), despite the presence of 10- to 20-fold more T cell in the PBPC inoculum. In the present study, we examined the effects of pretreatment of donors with G-CSF on GVHD, long-term engraftment, and lymphocyte reconstitution in a murine parent-->F1 model (B6.Ly-5a-->B6d2F1) using splenocytes as a source of peripheral progenitor cells. Recipients of splenocytes from G-CSF-treated donors experienced less mortality from acute GVHD and showed sustained weight gain by day 100 after transplantation. At that time, there was no histological evidence od GVHD in either liver or gut. Recipients of splenocytes from G-CSF-treated donors showed complete donor engraftment within 1 month, which was sustained until the end of the observation period. In contrast, recipients of T cell-depleted splenocytes showed slower donor engraftment and persistent donor/host chimerism. In addition, lymphocyte phenotype and function in mice receiving splenocytes from G-CSF-treated donors was significantly restored by day 100 after transplantation. Thus, the use of G-CSF-mobilized PBPC may provide significant advantages to conventional BMT by reducing GVHD without impairing long-term engraftment and immunologic reconstruction.

The immunopathophysiology of acute graft-versus-host-disease.

The major complication after allogeneic bone marrow transplantation (BMT) is the development of graft-versus-host-disease (GVHD). This disease is initiated during the conditioning of the recipient, when host tissues are damaged. During the afferent phase of the disease, alloreactive donor T cells recognize foreign major and minor histocompatibility antigens of host tissues. The efferent phase includes activation of inflammatory effector cells as well as the secretion of cytopathic molecules which induce pathology in skin, gastrointestinal tract, liver, lung, and the immune system. Substantial experimental and clinical evidence now indicates a central role of cytokines in the immunopathophysiology of acute GVHD, which forms the basis of this review. The balance between cytokines released by T helper 1 (Th1) cells (interleukin 2, interferon-gamma) or by T helper 2 (Th2) cells (interleukin 4, interleukin 10) after allogeneic BMT is hypothesized to govern the extent of the systemic inflammatory response. Because Th2 cytokines can inhibit the production of proinflammatory cytokines such as interleukin 1 and tumor necrosis factor-alpha, a Th1-->Th2 shift in the initial response of donor T cells may interrupt the cytokine cascade and thus offer a new approach to the prevention and treatment of acute GVHD. Successful interventions to modify the response of donor T cells may obviate the need for T cell depletion and thereby avoid the increased risk of relapse of malignancy and impairment of donor cell engraftment.

Interferon-gamma suppresses T-cell proliferation to mitogen via the nitric oxide pathway during experimental acute graft-versus-host disease.

The development of graft-versus-host disease (GVHD) is associated with long-lasting and profound deficits in immune function that lead to increased morbidity and mortality after bone marrow transplantation (BMT). We investigated a mechanism of T-cell immunodeficiency in response to mitogen or alloantigen in an experimental model of acute GVHD by analyzing the roles of two immunosuppressive moieties: interferon gamma (IFN-gamma) and nitric oxide (NO). Splenocytes from mice with GVHD did not proliferate either to the T-cell mitogen, concanavalin A (Con A), or to host alloantigens, but only mitogen-activated cultures produced increased levels of NO. The abrogation of NO synthesis with LG-mono-methyl-arginine (NMMA) restored mitogen-induced proliferation but not the response to host antigens. The mechanism of impared proliferation to mitogen was dependent on IFN-gamma because blockade of this cytokine in culture inhibited NO production and restored proliferation to Con A to levels similar to those in transplanted control mice without GVHD. NMMA did not substantially reduce IFN-gamma levels, demonstrating that NO acted distally to IFN-gamma in the pathway of immunosuppression in response to mitogen. Furthermore, the prevention of IFN-gamma production in vivo after allogeneic BMT, by transplantation of polarized type 2 donor T cells (secreting interleukin-4 but not IFN-gamma), also prevented NO production and restored splenocyte responses to mitogen. Our data demonstrate the existence of NO-dependent and NO-independent pathways involved in suppression of T-cell proliferation during acute GVHD. Excess NO synthesis appears to be one mechanism by which IFN-gamma induces immunodeficiency after allogeneic BMT.

TCR-beta transgenic mice fail to mediate a GVHR due to defects of allorecognition and subsequent IL-2 generation.

All T cells of TCR-beta transgenic mice bear a single TCR-beta chain and consequently the diversity of the TCR may be reduced by as much as one million-fold. Despite this limited diversity, many measures of lymphocyte function in these mice are normal. We have previously demonstrated that lymphoid cells from TCR-beta mice are unable to mediate an intense graft-versus-host response (GVHR). In order to investigate the mechanism of this hyporesponsiveness, we studied in vivo allorecognition in diverse strains of TCR-beta mice. All tested strains of TCR-beta mice failed to mediate a substantial GVHR across multiple H-2 barriers. In addition, mixtures of cells from several strains of TCR-beta mice only generated mild GVHRs. Sensitive tests of in vitro allorecognition show that lymphoid cells from TCR-beta mice respond less vigorously to alloantigen as measured both by decreased proliferation and decreased IL-2 production in a MLR. In addition, cells from TCR-beta mice fail to use exogenous IL-2 appropriately in their response to alloantigen. We conclude that the fixed TCR-beta chain causes a defective response to alloantigen, which is measured as decreased IL-2 generation and utilization, and that this abnormality results in a decreased GVHR.

Suppression of B-cell proliferation to lipopolysaccharide is mediated through induction of the nitric oxide pathway by tumor necrosis factor-alpha in mice with acute graft-versus-host disease.

Graft-versus-host disease (GVHD) is associated with impaired B-cell responses. We investigated the mechanism of impaired proliferation of B cells in response to the mitogen lipopolysaccharide (LPS) by analyzing the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO), both of which have independently been described as important effector mechanisms in the pathogenesis of acute GVHD. A threefold decrease of mature surface Ig-positive (slg+) B cells was observed in GVHD spleens isolated 2 weeks after transplant. However, proliferation of these cells in response to LPS was suppressed by more than 35-fold. Activated GVHD splenocytes secreted large amounts of TNF-alpha and NO in culture. Neutralization of TNF-alpha with anti-TNF-alpha antibody (Ab) both abrogated NO production and restored LPS-induced proliferation of B cells to levels found in non-GVHD control mice. The specific inhibition of NO synthesis with LG-monomethyl-arginine (NMMA) restored splenocyte responses but did not significantly reduce TNF-alpha levels, showing that TNF-alpha per se did not cause immunosuppression. These data show that, during GVHD, induction of the NO pathway is an important mechanism that mediates B-cell hyporesponsiveness to LPS and that this pathway is induced by TNF-alpha.

Dysregulation of cytokines during graft-versus-host disease.

Graft-versus-host disease (GVHD) remains a major complication following allogeneic stem cell transplantation. It is mediated by alloreactive donor T cells recognizing histocompatibility antigens of the host, and ex vivo depletion of these cells from the graft has been used as prophylaxis. This, however, carries increased risk of graft rejection, disease relapse, and impaired immune reconstitution. It now appears that GVHD may be primarily mediated by cytokines. A three-step hypothesis for the involvement of cytokines in the pathophysiology of acute and chronic GVHD is presented, with emphasis on the role of Th1 and Th2 T cell subsets.

Graft-versus-host disease and the Th1/Th2 paradigm.

Graft-versus-host disease (GVHD) is the major complication after allogeneic bone marrow transplantation (BMT) and is initiated by alloreactive donor T cells recognizing foreign histocompatibility antigens of the host. There is now substantial experimental and clinical evidence to implicate a dysregulation of cytokine networks as a primary cause for the induction and maintenance of GVHD. In this article, current knowledge of the involvement of cytokines in GVHD is reviewed. The balance between type 1 cytokines (interleukin-2, interferon-gamma) and type 2 cytokines (interleukin-4, interleukin-10) is hypothesized to govern the extent to which a cell-mediated immune response and a systemic inflammatory response develop after allogeneic BMT. Because type 2 cytokines can inhibit the production of the proinflammatory cytokines interleukin-1 and tumor necrosis factor-alpha, a type 1 to type 2 shift in the initial response of donor T cells to host alloantigens may interrupt the cytokine cascade after allogeneic BMT and may offer a new approach to the prevention and treatment of acute GVHD. Interventions to specifically eliminate or modify the response of donor T cells to alloantigens in order to reduce GVHD may obviate the need for T cell depletion in clinical BMT and thus avoid the increased risk of relapse of malignancy and impairment of donor cell engraftment.