The origin of IgG production and homogeneous IgG components after allogeneic bone marrow transplantation

Pediatric recipients (n = 25) of an allogeneic bone marrow (BM) graft were selected on the basis of informative IgG allotype (Gm) differences between the BM donor and the recipient. To investigate the kinetics of the appearance of IgG of donor origin and the disappearance of IgG of recipient origin, G1m and G2m allotype levels were quantified in sera obtained at regular intervals between 3 months and 5 years after BM transplantation (BMT). For this quantification, a dot immunobinding assay (DIBA) has been developed. In 19 of 22 informative recipients, the Gm allotype distribution had reached the range of values expected on the basis of the Gm phenotype of the donor within 6 months after BMT. Remarkably, IgG of recipient origin persisted in 15 of 18 informative recipients until last follow up, ie, for several years after BMT. In addition to the origin of total IgG production, the origin of homogeneous IgG components (H-IgG) appearing after BMT was investigated. H-IgG of donor origin could be detected as early as 3 weeks after BMT, but also H-IgG of recipient origin were present in 8 of 13 informative recipients for a period of up to 1 year after BMT. We conclude that host-type IgG-producing cells were not eradicated by the (myeloablative) conditioning regimen and persisted in a high number of graft recipients. It is our hypothesis that lack of graft-versus-host disease (GVHD) in the majority of these recipients results in the persistence of IgG-producing cells of host origin. These observations may be relevant for the evaluation of patients who received allogeneic BMT for the treatment of multiple myeloma.     

Increased presence of anti-HLA antibodies early after allogeneic granulocyte colony-stimulating factor-mobilized peripheral blood hematopoietic stem cell transplantation compared with bone marrow transplantation

We have recently shown that the use of allogeneic granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood hematopoietic stem cell transplantation (PBHSCT), as compared with bone marrow transplantation (BMT), is associated with increased titers of antibodies (Abs) directed against red blood cell ABO antigens. To further evaluate the influence of a G-CSF-mobilized PBHSCT graft on alloimmune Ab responses, we examined the frequency of anti-HLA Abs after transplantation in the setting of the same randomized study, comparing PBHSCT with BMT in adults. Anti-HLA Ab presence was determined by complement-dependent cytotoxicity assay (CDC) and flow cytometry in the recipient before and 30 days after transplantation as well as in the donor before graft donation. The use of PBHSCT was significantly associated with increased detection of anti-HLA immunoglobulin G (IgG) Abs early after transplantation as evidenced by flow cytometry (11 of 24 versus 4 of 27 transplant recipients, P =.03) and, less so, by CDC (5 of 24 versus 1 of 27 transplant recipients, P =.09). The difference between PBHSCT and BMT was further heightened when analysis was restricted to anti-HLA IgG Ab-negative donor/recipient pairs. In such a setting, early anti-HLA Ab was never detected after BMT but was repeatedly detected after PBHSCT (flow cytometry, 6 of 18 versus 0 of 17 transplant recipients, P =.02; CDC, 4 of 23 versus 0 of 26 transplant recipients, P =.04). Importantly, the PBHSCT-associated increase in anti-HLA Ab detection was observed despite a reduction in the median number of platelet-transfusion episodes per patient in PBHSC transplant versus BM transplant recipients (3 platelet-transfusion episodes [range, 1-21] in PBHSCT group vs 6 platelet-transfusion episodes [range, 3-33] in the BMT group; P =.02). In conclusion, this study strongly suggests that G-CSF-mobilized PBHSCT results in an increased incidence of circulating anti-HLA Abs and further confirms that the use of such a graft alters alloimmune Ab responses.     

Revaccination of bone marrow transplant recipients: a review of current practices in Australia

Background: Vaccination following bone marrow transplant (BMT) is an important part of ongoing care and disease prevention. The aim of the study was to investigate vaccination procedures in BMT recipients and identify what systems are in place throughout Australia to remind and alert patients concerning their need for vaccination.
Methods: Questionnaires were sent to haematologists managing BMT recipients in Australia to examine post-BMT vaccination practices in hospitals and outpatient clinics. Questionnaires were also sent to BMT recipients in New South Wales, who had their transplants (either allogeneic or autologous) in the past 5 years to determine what vaccinations they had received and what vaccination reminder systems had been used.
Results: Vaccine recommendations and practices by BMT physicians showed little consensus. They also differed greatly between autologous and allogeneic transplant recipients. Only just more than half of the physicians had an effective reminder system in place and only 12 of 34 patients had received vaccination reminders. One-third of all patients were not aware of any need for revaccination.
Conclusion: The disparity in physician practice regarding revaccination is significant and may reflect the lack of data available regarding efficacy of revaccination in this setting and/or a lack of knowledge about recommendations. Because of this, a national immunization schedule for post-BMT patients founded on evidence-based studies is required to provide optimal patient care. The lack of effective follow up and reminder systems ensuring patient completion of vaccination schedules is also an area needing improvement.     

Increased expression of Fas (APO-1, CD95) on CD34+ haematopoietic progenitor cells after allogeneic bone marrow transplantation

Up-regulation of Fas/APO-1 (CD95) on haematopoietic progenitors and Fas-mediated apoptosis have been suggested to occur in a possible pathological mechanism in some bone marrow failure syndromes. We examined the expression of Fas antigen and susceptibility to Fas-mediated suppression of donor-derived haematopoietic cells of allogeneic bone marrow transplantation (BMT) recipients. Cytofluorometric analysis revealed low expression of Fas on CD34+ bone marrow cells from marrow donors or healthy controls. However, significantly higher expression of Fas antigen was observed on CD34+ bone marrow cells of BMT recipients, in whom engraftment of donor bone marrow (BM) cells was confirmed. The addition of an agonistic anti-Fas antibody (Ab) (CH-11) to haematopoietic stem cell culture of BM cells more strongly suppressed colony formation from granulocyte-macrophage colony-forming units (GM-CFU) and erythroid burst-forming units (BFU-E) after BMT. Pretreatment by blocking anti-Fas Ab (ZB4) abrogated the Fas-mediated GM-CFU and BFU-E suppression. Purified marrow CD34+ cells from BMT recipients were also susceptible to the Fas-mediated colony suppression. Thus, donor-derived CD34+ haematopoietic cells increased their expression of Fas antigen and were susceptible to Fas-mediated haematopoietic suppression. These findings provide new insight for understanding the haematological condition after BMT.     

Abnormal T cell-dependent B-cell responses in SCID mice receiving allogeneic bone marrow in utero. Severe combined immune deficiency

In allogeneic hematopoietic stem cell transplant recipients, restoration of humoral immunity is delayed and can remain impaired for years. In many severe combined immune deficiency (SCID) patients given haploidentical bone marrow (BM), lesions in humoral immunity are exacerbated by poor engraftment of donor B cells. The nature of these defects is important to understand as they render patients susceptible to infection. Previous work in mice suggested that in utero transplantation (IUT) of allogeneic BM might offer several advantages for the correction of primary immune deficiencies. In SCID mice given fully allogeneic BM in utero, the lymphoid compartment was restored with minimal evidence of graft-versus-host disease (GVHD). The present report examines B-cell reconstitution and function in mice that have received allogeneic IUT. Results are compared with those of adult mice given total body irradiation (TBI) followed by transplantation with allogeneic BM. In addition to enumerating the various B-cell subsets present in BM, spleen, and peritoneal cavity (PC), B-cell competence was assessed by challenging mice with T cell-independent (TI) and T cell-dependent (TD) antigens. The results demonstrated that all B-cell subsets in the BM and periphery were restored in allogeneic IUT and TBI mice, as were antibody responses after TI challenge. Upon immunization with TD antigens, however, IUT and TBI mice exhibited suboptimal responses as measured by the capacity to isotype switch and generate germinal center (GC) B cells. Thus, although allogeneic BM transplantation results in complete recovery of the B-cell compartment, certain elements of the humoral response remain defective.     

Cytolytic function of clonable T cells after human bone marrow transplantation

We evaluated T-cell mediated lymphokine activated killer (LAK) function during the late (greater than 5 months) reconstitution phase after T cell-depleted allogeneic bone marrow transplantation (BMT) for hematologic malignancy. Since LAK cells are sustained by interleukin-2 (IL-2), we also investigated the ability of post-BMT T cells to produce IL-2. These functions were investigated at the clonal level. More than 200 T-cell clones from six long-term BMT recipients were generated and compared with 60 T-cell clones derived from two normal controls. Almost all the CD8+ clonal cultures from BMT recipients expressed cytolytic activity in a lectin-dependent cellular cytoxicity assay. Interestingly, a higher proportion of BMT recipient-derived cytolytic clones were able to mediate LAK activity in comparison with control clones (28% versus 4%, P less than .05). However, T-cell clones from BMT recipients, as opposed to control clones, were largely incapable of producing IL-2. Given the high proportions of post-BMT circulating CD8+ T cells, it appears that, in long-term BMT recipients, the precursors of nonspecific LAK effectors are present at above normal levels. However, their function may be defective in vivo due to poor IL-2 production.     

Reduced expression of vascular cell adhesion molecule-1 on bone marrow stromal cells isolated from marrow transplant recipients correlates with a reduced capacity to support human B lymphopoiesis in vitro

A common sequela to allogeneic or autologous bone marrow transplantation (BMT) is a delay in the reconstitution of a functional B-cell immune response. Therefore, we examined whether the posttransplant BM microenvironment is deficient in supporting the proliferation and/or differentiation of B-cell precursors. BM stromal cell cultures were established from patients who received allogeneic or autologous BMT for acute lymphoblastic leukemia, Hodgkin's disease, or non-Hodgkin's lymphoma. These cultures were then compared with normal donor BM stromal cell cultures for expression of adhesion molecules and the capacity to support the adhesion and interleukin-7 (IL-7)-dependent growth of normal B-cell precursors. Analysis of BM stromal cell cultures established from 28 BMT recipients showed a significantly reduced expression of cell surface vascular cell adhesion molecule-1 (VCAM-1/CD106), compared with normal donor BM stromal cells. Transplant BM stromal cell CD106 expression was responsive to regulatory cytokines in a manner qualitatively comparable with normal donor BM stromal cells. The level of B-cell precursor adhesion to transplant BM stromal cells correlated with the level of CD106 expression. Of 19 evaluable transplant BM stromal cell cultures, eight exhibited a reduced capacity to support the growth of CD19+/light chain- normal B-cell precursors. The capacity of transplant BM stromal cells to support B-cell precursor growth correlated with the level of CD106 expression, and the level of B-cell precursor adhesion. Our collective results may provide new mechanistic insight into why B-cell recovery is delayed post-BMT and underscore the importance of VCAM-1/CD106 in regulating B lymphopoiesis.     

Irradiated donor leukocytes promote engraftment of allogeneic bone marrow in major histocompatibility complex mismatched recipients without causing graft-versus-host disease.

Graft rejection in allogeneic bone marrow transplantation (BMT) can occur when donor and recipient are mismatched at one or more major histocompatibility complex (MHC) loci. Donor T cells can prevent graft rejection, but may cause fatal graft-versus-host disease (GVHD). We tested whether irradiation of allogeneic donor lymphocytes would preserve their graft-facilitating activity while inhibiting their potential for GVHD. Infusions of irradiated allogeneic T cells did not cause GVHD in MHC-mismatched SJL --> (SJL x C57BL6) F1, C57BL6 --> B10.RIII, and C57BL6 --> B10.BR mouse donor --> recipient BMT pairs. The 60-day survival among MHC-mismatched transplant recipients increased from 2% (BM alone) to up to 75% among recipients of BM plus irradiated allogeneic splenocytes. Optimal results were obtained using 50 x 10(6) to 75 x 10(6) irradiated donor splenocytes administered in multiple injections from day -1 to day +1. Recipients of an equal number of nonirradiated MHC-mismatched donor splenocytes uniformly died of acute GVHD. The graft facilitating activity of the irradiated allogeneic splenocytes was mediated by donor T cells. Irradiation to 7.5 Gy increased nuclear NFkappaB in T cells and their allospecific cytotoxicity. Irradiated T cells survived up to 3 days in the BM of MHC-mismatched recipients without proliferation. Recipients of irradiated allogeneic splenocytes and allogeneic BM had stable donor-derived hematopoiesis without a significant representation of donor splenocytes in the T-cell compartment. Irradiated allogeneic T cells thus represent a form of cellular immunotherapy with time-limited biologic activity in vivo that can facilitate allogeneic BMT without causing GVHD.     

Allogeneic MHC-mismatched activated natural killer cells administered after bone marrow transplantation provide a strong graft-versus-leukaemia effect in mice

Allogeneic lymphocytes administered with an unmanipulated bone marrow transplant provide a strong antileukaemic effect, the so-called graft-versus-leukaemia (GVL) effect. On the other hand, T-cell-mediated graft-versus-host-disease (GVHD) observed after transplantation of unmanipulated BM graft causes substantial morbidity and mortality. The aim of the present study was to determine the antileukaemic potential of enriched IL-2 activated NK cells administered 2 h after BMT. Balb/c (H-2^d) mice were given a dose of A20 (H-2^d, B-cell leukaemia) cells 2 d prior to lethal total body irradiation (TBI) and transplantation of either syngeneic or allogeneic anti-Thy1.2 (CD90) depleted bone marrow cells. Either syngeneic (Balb/c, H-2^d) or allogeneic (C57BL/6, H-2^b) enriched and IL-2 (200 U/ml for 24 h) activated NK cells were given 2 h after BMT. Injection of A20 leukaemia into normal Balb/c recipients led to death after a median of 14 d. A lethal dose of TBI followed by either syngeneic or allogeneic Thy1.2-depleted BMT resulted in a modest antileukaemic effect. The adoptive transfer of syngeneic enriched and IL-2 preincubated NK cells given at time of BMT exerted a significantly better GVL effect. However, the infusion of allogeneic enriched NK cells resulted in a stronger GVL effect. These results clearly demonstrate that allogeneic NK cells are superior to syngeneic NK cells in their potential to eradicate residual leukaemia cells after BMT without mediating clinical overt GVHD. This experimental setting may offer a strategy for treatment of haematological malignancies in a phase of minimal residual disease.     

Opsonophagocytic activity against Streptococcus pneumoniae type 19F in allogeneic BMT recipients before and after vaccination with pneumococcal polysaccharide vaccine

Opsonophagocytic activity (OPA) of pneumococcal polysaccharide (Pnc PS) antibodies in vitro, a measure of antibody functional activity, usually correlates with specific IgG antibody concentrations measured by an EIA method. In order to investigate the functional activity of specific Pnc PS antibodies, we determined IgG antibodies to Pnc PS type 19F by an EIA method and OPA against Pnc type 19F by a killing assay in a randomized study, where 23 adult allogeneic BMT recipients were vaccinated with Pnc PS vaccine at 8 months (early group) and 21 recipients at 20 months (late group) after transplantation. Serum samples drawn before BMT, before vaccination, and at 1 and 16 months after vaccination were available from 27, 35, 34 and 30 patients, respectively. The geometric mean anti-Pnc 19F concentrations were 4.3, 1.3, 1.6 and 1.3 microg/ml in the early group and 3.8, 0.9, 0.6 and 0.6 microg/ml in the late group before transplantation, before vaccination, and at 1 and 16 months after vaccination, respectively. OPA (titre > or = 8) was found in 10/27, 5/35, 5/34 and 6/30 patients before BMT, before vaccination, and at 1 and 16 months after vaccination, respectively. The specific IgG antibody concentration and OPA correlated with each other before BMT, and in the early group patients before and at 1 month after vaccination. The results demonstrate that after Pnc PS vaccination allogeneic BMT recipients have antibodies with low functional activity to a Pnc PS antigen associated with low specific IgG responses. There is a need to study new Pnc conjugate vaccines in multi-dose schedules for their capacity to elicit higher specific antibody concentrations with high OPA in BMT recipients.     

Costimulatory blockade of CD154-CD40 in combination with T-cell lymphodepletion results in prevention of allogeneic sensitization

Sensitization is a critical unresolved challenge in transplantation. We show for the first time that blockade of CD154 alone or combined with T-cell depletion prevents sensitization. Allogeneic skin grafts were rejected by recipients treated with anti-alphabeta T-cell receptor (TCR), anti-CD154, anti-OX40L, or anti-inducible costimulatory pathway (ICOS) mAb alone with a kinetic similar to untreated recipients. However, the production of anti-donor MHC antibody was prevented in mice treated with anti-CD154 mAb only, suggesting a specific role for the CD154-CD40 pathway in B-cell activation. The impairment of T cell-dependent B-cell responses by blocking CD154 occurs through inhibiting activation of T and B cells and secretion of IFN-gamma and IL-10. Combined treatment with both anti-CD154 and anti-alphabeta TCR abrogated antidonor antibody production and resulted in prolonged skin graft survival, suggesting the induction of both T- and B-cell tolerance with prevention of allogeneic sensitization. In addition, we show that the tolerance induced by combined treatment was nondeletional. Moreover, these sensitization-preventive strategies promote bone marrow engraftment in recipients previously exposed to donor alloantigen. These findings may be clinically relevant to prevent allosensitization with minimal toxicity and point to humoral immunity as playing a dominant role in alloreactivity in sensitized recipients.     

Tetravalent meningococcal polysaccharide vaccine is immunogenic in adult allogeneic BMT recipients

Forty-four adult BMT recipients transplanted from an HLA-identical sibling donor were randomized to receive meningococcal polysaccharide (Men PS) vaccine either 8 (early group; 22 patients) or 20 (late group; 22 patients) months after BMT. The geometric mean concentrations (GMC) of antibodies to serogroup A Neisseria meningitidis (Men A) and serogroup C Neisseria meningitidis (Men C), determined by an EIA method, decreased during the first 6 months after BMT but remained at a stable level thereafter. Before vaccination the GMCs of anti-Men A were 1.53 microg/ml and 1.61 microg/ml, but 1 month after vaccination they were significantly higher, 3.46 microg/ml and 6.39 microg/ml, in the early and late groups. The GMCs of anti-Men C increased from 0.37 microg/ml and 0.44 microg/ml before vaccination to 3.31 microg/ml and 4.62 microg/ml at 1 month after vaccination in the early and late groups, respectively. By 6 months after vaccination the GMCs of Men antibodies had decreased to levels of about 50% of those measured at 1 month after vaccination. Two-fold responses to Men A PS were seen in 52% and 74% and to Men C PS in 76% and 89% of the BMT recipients in the early and late groups, respectively. Chronic GVHD had no influence on the vaccination response. In the present study, Men PS vaccine induced good and equal antibody responses to Men A and Men C PSs in allogeneic BMT recipients regardless of timing after BMT. Vaccination against Neisseria meningitidis should be considered, especially in the event of travelling or military service > or = 8 months after BMT.     

Immunoglobulin E levels following allogeneic, autologous, and syngeneic bone marrow transplantation: an indirect association between hyperproduction and acute graft-v-host disease in allogeneic BMT

Markedly elevated serum IgE levels have been noted following allogeneic bone marrow transplantation (BMT) and have been correlated with graft-v- host disease (GVHD) in several studies. To investigate this phenomenon, we measured serum IgE levels in 387 allogeneic, 143 autologous, and 21 syngeneic BMT recipients before and at intervals after BMT. As a population, allogeneic BMT recipients displayed a biphasic elevation in IgE levels, with peak levels occurring either early (days 15 to 19) or late (days 80 to 89) posttransplant. Only in individuals in whom peak levels occurred early did IgE level correlate with liver disease, histological changes, and overall clinical stage of GVHD. The association of IgE elevation and GVHD does not appear to be direct since recipients of syngeneic (monozygotic twin) grafts had the highest incidence of IgE hyperresponsiveness as well as the highest absolute IgE levels. Similarly, 22 recipients of autologous marrow not treated with 4-hydroperoxycyclophosphamide had elevated IgE levels comparable to those seen in allogeneic graft recipients. We hypothesize that augmented IgE synthesis and its subsequent resolution is the natural consequence of immune reconstitution in the presence of potentially reaginic agents such as antibiotics and infectious agents. As such, IgE hyperresponsiveness in syngeneic graft recipients may reflect the maturational sequence of IgE regulatory elements in the absence of interference by GVHD, GVHD therapy, or minor histocompatibility disparities. The cell populations required for IgE response (T cells, B cells, and antigen-presenting cells) may be reconstituted in advance of the regulatory elements that limit IgE production in healthy subjects. Although this temporal relationship does not appear to hold in allogeneic BMT, the balance between positive and negative factors, which determines the rates of IgE synthesis and catabolism, may be altered by GVHD, infection, and liver dysfunction acting alone or in combination.     

Interleukin 18 preserves a perforin-dependent graft-versus-leukemia effect after allogeneic bone marrow transplantation

We have recently shown that early administration of interleukin 18 (IL-18) after bone marrow transplantation (BMT) attenuates acute graft-versus-host disease (GVHD) in a lethally irradiated parent into F1 (B6-->B6D2F1) BMT model. In this study, we investigated whether IL-18 can maintain graft-versus-leukemia (GVL) effect in this context. B6D2F1 mice received transplants of T-cell-depleted (TCD) bone marrow (BM) and splenic T cells from either syngeneic (H2(b/d)) or allogeneic B6 (H2(b)) donors. Recipient mice were treated with recombinant murine IL-18 or the control diluent. Initial studies demonstrated that IL-18 treatment did not affect the proliferative responses or the cytolytic effector functions of T cells after BMT. In subsequent experiments, animals also received host-type P815 mastocytoma cells at the time of BMT. All syngeneic BM transplant recipients died from leukemia by day 18. The allogeneic BM transplant recipients effectively rejected their leukemia regardless of treatment and IL-18 significantly reduced GVHD-related mortality. Examination of the cytotoxic mechanisms with perforin-deficient donor T cells demonstrated that perforin is critical for the GVL effect. Taken together these data demonstrate that IL-18 can attenuate acute GVHD without impairing the in vitro cytolytic function or the in vivo GVL activity after allogeneic BMT.     

Chronic GvHD decreases antiviral immune responses in allogeneic BMT

Chronic graft-versus-host disease (cGvHD) is associated with functional immunodeficiency and an increased risk of opportunistic infections in allogeneic bone marrow transplantation (BMT). We used a parent to F1 model of allogeneic BMT to test the hypothesis that cGvHD leads to impaired antigen-specific antiviral immunity and compared BM transplant recipients with cGvHD to control groups of allogeneic BM transplant recipients without GvHD. Mice with and without cGvHD received a nonlethal dose of murine cytomegalovirus (MCMV) +100 days after transplantation. Recipients with cGvHD had more weight loss and higher viral loads in the spleen and liver. MCMV infection led to greater than 25-fold expansion of donor spleen-derived MCMV peptide-specific tetramer-positive CD8(+) T cells in blood of transplant recipients with and without cGvHD, but mice with cGvHD had far fewer antigen-specific T cells in peripheral tissues and secondary lymphoid organs. The immunosuppression associated with cGvHD was confirmed by vaccinating transplant recipients with and without cGvHD using a recombinant Listeria expressing MCMV early protein (Lm-MCMV). Secondary adoptive transfer of lymphocytes from donor mice with or without cGvHD into lymphopenic congenic recipients showed that cGvHD impaired tissue-specific homing of antigen-specific T cells. These results indicate that cGvHD causes an intrinsic immunosuppression and explain, in part, the functional immunodeficiency in allogeneic transplant recipients.     

Defective antifungal T-helper 1 (TH1) immunity in a murine model of allogeneic T-cell-depleted bone marrow transplantation and its restoration by treatment with TH2 cytokine antagonists

Patients undergoing full haplotype-mismatched hematopoietic transplantations may experience severe intractable invasive fungal infections. To verify whether an imbalanced production of T-helper 1 (TH1) and TH2 cytokines may be responsible for susceptibility to fungal infections, C3H/HeJ (H-2(k)) recipient mice were lethally irradiated, received transplantations with T-cell-depleted allogeneic bone marrow (BM) cells from mice of H-2(d) haplotype, and were infected with Candida albicans. At different time-points after transplantation, mice were assessed for pattern of TH cytokine production and susceptibility to infection. The results show that a long-term, donor-type chimerism was achieved as early as 2 weeks after BM transplantation (BMT), at the time when high-level production of TH2 cytokines (interleukin-4 [IL-4] and IL-10) and impaired production of TH1 cytokines (interferon-gamma [IFN-gamma] and IL-12] were observed. At this time, mice were highly susceptible to both disseminated and mucosal infections, as indicated by decreased survival, uncontrolled fungal growth, and failure to develop protective TH1 immunity. However, a predominant production of TH1 cytokines was observed by week 5 after BMT, at the time when mice developed donor-type protective TH1 responses and were resistant to infections. Therapeutic ablation of IL-4 or IL-10 greatly increased resistance to candidiasis. These results indicate that a dysregulated production of TH cytokines occurs in mice undergoing T-cell-depleted allogeneic BMT. The transient predominant production of TH2 cytokines over that of IL-12 impaired the ability of mice to develop antifungal TH1 resistance, an activity that could be efficiently restored upon treatment with TH2 cytokine antagonists.     

Nonmyeloablative allogeneic bone marrow transplantation as immunotherapy for hematologic malignancies and metastatic solid tumors in preclinical models

OBJECTIVE: We previously demonstrated that a combination of mild total lymphoid irradiation (TLI) with selective depletion of the host's donor-reactive cells allows for stable and graft-vs-host disease (GVHD)-free engraftment of allogeneic bone marrow (BM). In this study, we investigated the efficacy of this nonmyeloablative strategy for BM transplantation (BMT) as immunotherapy for minimal residual disease. MATERIALS AND METHODS: BALB/c mice inoculated with leukemia (BCL1) or breast carcinoma (4T1) cells were conditioned for BMT with TLI (200 cGy) followed by priming with donor (C57BL/6) BM cells on day 1, and by injection with 200 mg/kg cyclophosphamide on day 2. After conditioning (day 3), recipients were transplanted with BM cells from the same donor. Treated animals were monitored for 230 days for survival, development of leukemia/solid tumor, and GVHD. RESULTS: BMT converted the mice to complete chimeras and prevented development of leukemia in 90% of recipients and locally growing breast carcinoma in 40% of the mice. Immunization of donors of the second BM with 4T1 cells prevented development of breast carcinoma in 80% of 4T1 inoculated mice. Fewer animals treated for malignancy by nonmyeloablative BMT died of GVHD than those treated by myeloablative BMT. However, late GVHD-related mortality in mice treated for leukemia was higher than after nonmyeloablative BMT to naive recipients (p < 0.00001). Infusion of host-type anti-donor immune lymphocytes 8 days after BMT improved the survival of recipients treated for leukemia without affecting engraftment and the graft-vs-leukemia potential of donor BM. CONCLUSIONS: Effective eradication of malignant cells can be achieved following allogeneic BMT after nonmyeloablative conditioning.     

Adoptive immunotherapy of BCR-ABL-induced chronic myeloid leukemia-like myeloproliferative disease in a murine model

Donor leukocyte infusion (DLI) can induce graft-versus-leukemia (GvL) reactions in patients with chronic myeloid leukemia (CML) relapsing after allogeneic bone marrow transplantation (BMT), but the mechanisms of the antileukemic effect of DLI are unknown, and the procedure is complicated by graft-versus-host disease (GvHD) and graft failure. Here, we adapted a murine retroviral BMT model of Philadelphia(+) leukemia by combining allogeneic bone marrow (BM) from C57Bl/6 (H-2(b)) mice with BCR-ABL-transduced Balb/c (H-2(d)) BM, inducing mixed chimerism and myeloproliferative disease in recipients resembling relapse of CML following allogeneic BMT. Infusions of allogeneic splenocytes eliminated BCR-ABL-induced CML-like disease in the majority of mixed chimeras, with significant GvL effects mediated by both CD4(+) and CD4(-) cells. BCR-ABL-induced acute B-lymphoblastic leukemia was also eradicated by DLI in major histocompatibility complex (MHC)-mismatched chimeras. Most DLI-treated mice converted to full allogeneic chimerism but succumbed frequently to GvHD or graft failure. When MHC-matched B10.D2 (H-2(d)) mice were the allogeneic donors, CML-like disease was more resistant to DLI. These results suggest that depletion of CD8(+) cells from DLI could impair GvL against CML, while increased MHC disparity between donor and recipient may improve the responsiveness of Philadelphia(+) B-lymphoblastic leukemia to DLI.     

Impaired B-cell reconstitution in lymphoma patients undergoing allogeneic HSCT: an effect of pretreatment with rituximab?

Allogeneic hematopoietic SCT (HSCT) is increasingly considered an option in refractory or relapsing lymphoma. Today, most patients with B-cell lymphoma are treated with the monoclonal anti-CD20 antibody rituximab before HSCT. We hypothesized that prior therapy with rituximab might alter immune reconstitution after allogeneic transplantation due to in vivo depletion of B cells at the time of graft infusion. We studied B-cell immune reconstitution in 12 patients with lymphoma receiving rituximab 1-12 months before HSCT. Compared to an age- and sex-matched population of patients transplanted for myeloid malignancies, lymphoma patients with rituximab pretreatment showed significantly reduced B-cell counts at time of HSCT at +3, +6 and +12 months; B-cell counts reached values comparable to controls only 24 months after HSCT. In parallel, levels of immunoglobulins were markedly reduced for up to 2 years post transplant in patients with prior rituximab treatment. Two patients suffered from severe late bacterial infections to which the impaired humoral immunity may have contributed. In contrast, T- and NK-cell reconstitution was not different compared to control patients.In conclusion, B-cell reconstitution can be significantly delayed in allogeneic HSCT recipients with prior rituximab treatment. Rituximab appears to have clinical consequences beyond the immediate early post-transplant period.     

Allogeneic peripheral blood stem cell versus bone marrow transplantations: CD34+ cell mobilisation,graft composition,immune reconstitution and clinical outcome

The use of peripheral blood, after G-CSF mobilization, as a source of allogeneic hematopoietic stem cells is being increasingly considered. Some significant data were given by 3 large randomized trials comparing bone marrow (BM) and peripheral blood stem cells (PBSC). Numbers of CD34+ and CD3+ harvested cells were significantly higher in PBSCT group and the hematopoietic recovery was significantly faster after PBSCT. While the American trial showed significant increased rates of acute GVHD and an higher survival and disease-free survival in favour of PBSC, the 2 other studies showed a significant increase of chronic GVHD after PBSCT. The French group investigated whether there was a correlation between cellular composition of PBSC and outcome after transplant. Neither hematological recovery, acute or chronic GVHD nor disease relapse, were significantly associated with CD3+ cell doses. However, high CD34+ cell doses (>8.3×10⁶/Kg) were associated with faster hematopoietic recovery and higher probability of extensive cGVHD at 5 years and DFS was significantly higher in patients receiving low CD34+ cell dose. In parallel, in this study, we evaluated and compared different immune parameters in the graft and after transplantation. Absolute values of mononuclear cells/kg were significantly higher in PBSC grafts than in BM grafts. Analysis of CD25, CD95, HLA-DR and CD45RA expression showed that PBSC grafts T cells exhibited a lower activation level. We found no preferential G-CSF-induced mobilisation of so-called “suppressive” CD3+ cells. In contrast, G-CSF reduced 2- to 3-fold the frequency of IFN-gamma-, IL-2- and TNF-alpha-secreting cells within the NK, NK-T and T-cell subsets and severely reduced the potential for IFN-gamma production at the single-cell level. Thirty days after transplantation, T-cell blood counts were 3-fold higher after PBSC. After PBSCT, T cells were less activated, and at day 30 CD4+, CD8+, CD45RA+ counts were correlated with the number of mononuclear cells infused with the graft, this was not observed after BMT. We showed that Anti-A and/or anti-B Ab titers were significantly increased in PBSCT vs BMT recipients at day 30 and mostly after minor ABO mismatch transplant. PBSCT were significantly associated with increased detection of anti-HLA antibodies early after transplantation. This difference was further increased when analysis was restricted to anti-HLA IgG Ab-negative donor/recipient pairs. The higher number of B cells in the PBSC could be associated with enhanced Ab production early after transplantation. Regarding mini-allotransplants, in France, in a retrospective analysis, we did not show any difference between PBSC and BM in term of disease response, chimerism and overall survival but a significant impact of number of CD34+ cells on early chimerism after transplant. This kind of tranplant might include donor lymphocyte infusion (DLI) strategy and we showed in our center that previous PBSC donation selectively impaired the yields of total MNC and total CD3+ cells recovery after donation but did not affect neither CD19+ nor CD3? CD56+ recoveries when compared to the cohort of BM donations. The use of G-CSF mobilised peripheral blood, as a source of allogeneic stem cells is increasingly considered, especially for mini-allotransplants which justify further studies to identify the different biological characteristics of this source and its clinical impact. *** DIRECT SUPPORT *** A00RC002 00004