Graft-versus-host Reaction Research Articles

Prevention of Graft-Versus-Host Disease in Mouse Model Using Anti-Mouse C5 Antibody.

Graft versus-host disease (GVHD) is a major cause of morbidity and mortality after bone marrow transplantation (BMT). An allogeneic GVH reaction is a response of donor lymphoid cells to host minor or major histocompatibility antigens. Donor T cells can be activated through the innate and the adaptive immune mechanisms. Donor B cells produce antibodies directed to host cells. These mechanisms may activate complement pathways. Thus, complement may have a crucial role in inflammation during a GVH reaction, but direct evidence for this has not been shown. In this study, we investigated the possibility of complement inhibitor, anti-mouse C5 antibody (BB5.1), to ameliorate the symptoms of GVHD using an acute GVHD mouse model: C57BL/6 (H2b) →BALB/c (H2d). One million T cells were injected together with 1 x 107 T-cell-depleted bone marrow (TCD BM) cells via tail vein into lethally irradiated BALB/c (8.5 Gy) recipients. Anti-mouse C5 antibody or its isotype matched control was administered intraperitoneally at a dose of 1 mg/mouse, 3 doses/week, for 4 weeks. Recipients were weighed weekly, and their survival was monitored daily. Average body weight of C5 antibody treated mice was 15.8 g at day 84 (19.2 g at day 0, N=12), whereas average weight of control mice was 13.3 g (19.2 g at day 0, N=12) (P=0.05, Student's t-test). Kaplan-Meier survival curves were also compared as shown in the Figure. Eight of 12 mice were alive at day 84 in the treated group, as compared to only 2 of 12 in the control group (P=0.03, Logrank test). A second experiment showed similar data. We, thus, observed the effect of anti-mouse C5 antibody to reduce the symptoms of GVHD using an acute GVHD mouse model. These results might open a new window for the prevention of acute GVHD. Further experiments are currently ongoing to clarify the exact mechanism between complement and GVHD. [Display omitted]

Eradication of B Cell Malignancies by Anti-3rd Party CTLs: A Novel Role for MHC Class I.

Induction of donor type chimerism under reduced intensity conditioning in the absence of GVHD represents a major challenge in transplantation biology. Despite their remarkable potent veto activity, CD8 CTLs could not be used to enhance engraftment due to their GVH reactivity. To address this problem we developed a new approach for the generation of human host-nonreactive CTLs, based on stimulation of donor CD8 T cells against 3rd party stimulators under IL-2 deprivation. A major attribute of such anti-3rd party CTLs reported recently by our group is their ability to eradicate pathological B-CLL cells. This B-CLL killing was shown to be independent of TCR recognition and it was found to be mediated both by autologous and by allogeneic anti-3rd party CTLs. In the present study we demonstrate that anti-3rd party CTLs can also efficiently eradicate primary mantle lymphoma cells and Burkitt and mantle cell-lines. Furthermore, the use of lymphoma cell lines has enabled to define several aspects of the mechanism leading to their apoptosis. Thus, we found that anti-3rd party CTLs avidly bind to target lymphoma cells and form stable aggregates within the first minutes of contact. Conjugate formation as well as the actual killing of the tumor cells were effectively inhibited by blocking antibodies against LFA1 or ICAM1 but not against ICAM3. Moreover, effective inhibition was attained only when applying anti-LFA1 to the CTLs and anti-ICAM1 to the tumor cells (100%±10% and 72%±7% inhibition, respectively) but not vice-versa. Given the role of CD8-MHC class I interaction in veto mechanisms, we sought to evaluate its role in the killing of lymphoma cells by human anti-3rd party CTLs. Indeed, in 10 experiments survival of target lymphoma cells was increased from 50%±5% to 82%±1% upon addition of anti HLA-ABC Ab to the incubation with anti 3rd party CTLs. A similar survival increase to 77.5%±1% was found upon the addition of anti-CD8 Ab, suggesting that CD8 binding to MHC class I constant region is required for lymphoma cell killing. The important role of MHC class I was further established by comparing CTL-mediated killing of Daudi lymphoma cells lacking cell surface MHC class I expression and Daudi cells with reconstituted surface MHC class I due to stable β2-microglobulin overexpression (β2m-Daudi). Thus in contrast to the poor killing of Daudi cells which did not exceed 20%, the β2m-Daudi cell killing was comparable to that exhibited with burkitt lymphoma line expressing MHC class1 (56%±1% and 63%±3%, respectively in 4 experiments). Importantly, FACS analysis showed that, MHC I expression did not influence conjugate formation, but was rather involved in the induction of apoptosis (annexin V). Furthermore, a similar pattern was also found in vivo when β2m-Daudi or Daudi cells were infused into the peritoneum of SCID mice in the presence or absence of a 2.5-fold excess of anti 3rd party CTLs. In 3 experiments, FACS analysis of the cells recovered from the peritoneum showed that the CTLs killed 91% of the β2m-Daudi cells as opposed to 13% killing of the Daudi cells. Taken together, our results suggest that TCR independent killing of B cell malignancies by anti-3rd party CTLs is mediated via a rapid adhesion through ICAM1-LFA1 binding, followed by slow induction of apoptosis upon a critical interaction between CD8 on the CTL and MHC class I on the tumor cell.

Unmanipulated HLA 2-3 antigen–mismatched (haploidentical) bone marrow transplantation using only pharmacological GVHD prophylaxis

The incidence of severe graft-vs-host disease (GVHD) in unmanipulated human leukocyte antigen (HLA) 2-3 antigen-mismatched bone marrow transplantation (BMT) using cyclosporine and methotrexate as GVHD prophylaxis is 80% to 90%. We investigated whether pharmacological GVHD prophylaxis consisting of four drugs, including a steroid, effectively suppressed GVHD in this transplantation setting. Thirty patients who had hematologic malignancies at an advanced stage or with poor prognosis underwent allogeneic BMT using a myeloablative preconditioning regimen consisting of cyclophosphamide (60 mg/kg x 2), total body irradiation (8-10 Gy), and fludarabine (30 mg/m(2) x 4) with or without cytosine arabinoside (2 g/m(2) x 4), and GVHD prophylaxis consisting of a combination of tacrolimus, methotrexate, mycophenolate mofetil, and methylprednisone (2 mg/kg). Early therapeutic intervention for GVH reaction or grade I GVHD was performed, and steroid was slowly tapered. All patients achieved donor-type engraftment. Neutrophil (>0.5 x 10(9)/L) and platelet (>20 x 10(9)/L) engraftment was achieved on day 13 and on day 30, respectively. Seventeen patients (56.7%) had no GVHD. Eleven patients (36.7%) developed grade II-III acute GVHD. Seven patients (23.3%) died of transplant-related toxicity, including fungal or viral infections and thrombotic microangiopathy, and four patients died of disease progression. Estimated relapse rate at 3 years was only 20.9%. The probability of survival at 3 years was 49.9%. These data suggest that, in unmanipulated HLA-haploidentical allogeneic BMT, this GVHD prophylactic regimen, which includes methylprednisolone 2 mg/kg, and early therapeutic intervention for GVH reaction suppress the incidence of severe GVHD to an acceptable level, while preserving the graft-vs-leukemia effect.

Development ofNovel Compounds to Treat Autoimmune and Inflammatory Diseases and Graft Versus Host Reactions

Recently, several new classes of agents were developed to treat patients with malignant diseases. This progress has been based on the advances made in our understanding of critical pathways involved in tumor development and growth. Dysregulated processes leading to uncontrolled regulation of proliferation, cell cycle progression, angiogenesis and apoptosis have provided rational targets for novel therapies. Compounds inhibiting protein phosphorylation and signal transduction like tyrosine kinase inhibitors and inhibitors of proteasomal degradation have demonstrated promising results and were approved for the treatment of patients with malignant diseases. However, based on in vitro and in vivo studies, there is now an emerging evidence that these agents can affect the function and differentiation of normal, non-malignant cells like dendritic cells or T lymphocytes, resulting in immunosuppression. In our review we present recent data on the immune regulatory effects of tyrosine kinase inhibitors like imatinib that is approved to treat chronic myeloid leukemias, or inhibitors of FLT3, currently used to treat acute leukemias, as well as proteasome inhibitors and peroxisome proliferator-activated receptor agonists and discuss their possible role and application in the treatment of autoimmune and graft versus host disease.

DC homeostasis in hematopoietic stem cell transplantation

Hematopoietic stem cell transplantation is an important experimental tool and therapeutic modality. Its efficacy and toxicity are both linked to a GvH reaction that is initiated by donor T cells recognizing recipient APC, of which DC are the most potent. In most tissues recipient DC are replaced after transplantation because they turnover rapidly from BM-derived precursors. However, in a number of sites, notably the skin, recipient DC may persist and even self-renew for many months after transplantation. Understanding the homeostasis of different APC populations and how they are related to the induction of alloreactivity may help to improve the therapeutic benefit of transplantation.

Integrated reticuloendotheliosis virus genomic sequences in fowlpox virus.

Abstract Fowlpox is a common viral disease in domestic poultry, with a worldwide distribution. The negative impacts of the disease are reduction in egg production and retarded growth in younger birds. Vaccination with live vaccines has long been used for the control of the disease in the poultry industry and economic losses have been minimized. However, in recent years, this disease re-emerged as a new threat to poultry production, particularly in vaccinated flocks. Reticuloendotheliosis virus (REV) sequences were detected in the genomes of the fowlpox virus (FPV) field isolates causing the disease outbreaks and subsequently in archival virus isolates. It is interesting that this integration event occurred over 50 years ago. It has been found that most fowlpox field viruses contain REV provirus, while vaccine strains (except FPV S strain, an obsolete Australian vaccine strain) contain only remnants of REV long terminal repeats (LTRs). The integrated REV provirus appears to be infectious, as antibody responses to REV have been detected in infected birds. The presence of REV provirus in FPV field isolates has resulted in enhanced pathogenicity of the virus. In addition, clinical signs associated with REV, including runting disease syndrome and immunosuppression, have been demonstrated in young birds. The retention of the REV provirus may have resulted in modification of host gene (FPV genome) activities such as gene activation or inactivation, giving rise to alteration of the viral phenotype. This may have contributed to antigenic variation, leading to inadequate protection in vaccinated flocks.

Prevention of Graft-Versus-Host Disease by Selective Depletion of Alloreactive T Cells.

OBJECTIVE: Mismatched allogeneic hematopoietic cell transplantation (alloHCT) carries a high risk of life-threatening graft-versus-host disease (GVHD) due to activation of donor T cells by antigens present on host cells. Removal of donor mature T cells can prevent GVHD but leads to an increased incidence of opportunistic infections and disease relapse. This study aims to selectively deplete host-reactive donor T cells responsible for GVHD while preserving T cells with anti-tumor and anti-viral effects.METHODS: We utilized a photosensitizer, 4,5-dibromorhodamine-methyl ester (TH9402, Celmed Biosciences Inc., Saint-Laurent, Canada), in an ex vivo photodynamic purging (PDP) process to specifically eradicate host-reactive T cells. Donor T cells with anti-host specificity were identified in a unidirectional mixed lymphocyte culture (MLC) where they were activated and became proliferating. TH9402 is taken up by all cells and extruded out of the cell by P-glycoprotein (Pgp) in non-activated cells.However, due to inactivation of Pgp in activated T cells, TH9402 is retained in the mitochondria. Upon exposure to 514 nm light in the Theralux™ device (Celmed), it becomes extremely cytotoxic resulting in cell death. In this study, after treatment with various concentrations of TH9402, the cells were exposed to light for the elimination of alloreactive T cells. The efficiency of allodepletion was assessed by Granzyme B (GrB) assay. T-cell proliferation assays were used to demonstrate the preservation of anti-tumor and anti-viral effects. Finally, the skin explant assay, an in vitro model of GVHD, was utilized to examine the efficacy of the PDP treatment in the removal of alloreactive T cells responsible for GVHD. The parameters of the PDP treatment were optimized for use in subsequent clinical studies.RESULTS: After 72-hour MLC, optimal proliferative response was obtained at a responder: stimulator ratio of 1:1. Activated T cells expressed high level of activation markers such as CD25 and CD69. After the PDP treatment with 20μM of TH9402, alloreactive T cells were consistently depleted by more than 2 logs (Figure 1). Moreover, the PDP treatment did not significantly affect anti-tumor and anti-viral effects as evidenced by responses to third-party stimulators (Figure 2A), cytomegalovirus (CMV) (Figure 2B) and Candida antigens. Most importantly, co-culture of recipient's skin with PDP-treated cells showed a reduction of graft-versus-host reaction (GVHR) in a TH9402-dose dependent manner. The PDP treatment with 20μM of TH9402 completely abolished GVHR in a skin explant assay.CONCLUSIONS: The PDP treatment can effectively remove donor T cells responsible for GVHD while preserve T cells with anti-tumor and anti-viral effects. These preclinical results provide a basis for initiating a clinical trial to assess the feasibility and efficacy of infusing PDP-treated donor T cells to alloHCT recipient in order to augment anti-tumor and anti-pathogen effects without causing GVHD. [Display omitted] [Display omitted]

Partly MHC Matched Allogeneic Tumor Specific T Cells Mediate Tumor Regression without Inducing GVHD in Immunosuppressed Host.

Adoptive cell transfer (ACT) immunotherapy following lymphodepleting conditioning is a promising strategy for the treatment of metastatic solid tumors, however the difficulties in generating autologous tumor specific lymphocytes for every patient has significantly limited its applications. Allogeneic partly matched tumor specific T cells could be used for patients in whom autologous cells are not available, however their rapid rejection by the host restricts this approach. When CD8+ pmel-1 T cells from B6-BALB-C F1 (b/d) were transferred into B16 tumor-bearing B6 mice (b/b) or into a different F1 (b/k), these tumor-specific T cells were rapidly rejected, and had no impact on the tumor regression. Here we show that following myeloablative conditioning, the adoptive transfer of allogeneic, major histocompatibility mismatched tumor-specific T lymphocytes resulted in the regression of large vascularized tumors. The ability of adoptively transferred allogeneic T cells to mediate tumor regression was directly proportional to the dose of irradiation given prior T cell transfer which also correlated with the in vivo survival of the transferred cells. At the highest irradiation dose used (i.e. 11 Gy) allogeneic T cells could survive for as long as 4 weeks and their efficacy was comparable to syngeneic tumor-reactive T cells. In addition we found that the risk of inducing a graft versus host (GVH) reaction was minor when the specificity of transferred TCR is confined. In fact co-transfer of transgenic cells and small amounts of open repertoire T cells (2*10e4) able to react with the host did not result in any measurable toxicity whereas co transfer of greater quantities (2*10e5 or more) could cause fatal GVHD effect. Interestingly GVH reaction was associated with an improved tumor treatment, though this effect was transient as most of the animals succumbed to GVHD. Here we demonstrate that allogeneic T cells might represent an important tool in cancer immunotherapy allowing treatment of patients for whom it is not possible to obtain autologous cells. Furthermore the possible synergy between tumor specific allogeneic T cells and GVH effect could be exploited in bone marrow transplant protocols.

Vaccination with the WT-1 126-134 Peptide in Patients with Acute Myeloid Leukemia after Allogenic Stem Cell Transplantation.

The induction of a graft versus leukaemia (GvL) effect following allogenic hematopoietic stem cell transplantation (HSCT) is critical for the outcome in patients with hematological malignancies. Tumor-specific antigens as WT-1 are expressed on leukemic blasts and are major targets of the GvL effect. Therefore the generation of a WT-1 specific immune response through peptide vaccination could enhance the antileukemic effects after HSCT. In a phase I study five HLA A*0201 positive patients with high-risk AML (median age 64 yrs, range 33–67yrs) were vaccinated with the WT-1 derived peptide RMFPNAPYL (126–134) beginning at day 21 after HSCT. The vaccination protocol consisted in four biweekly vaccinations with the peptide in a dose of 0,2mg administered i.d. and s.c. with 1mg of keyhole limpet hemocyanin (KLH) as adjuvant. Concomitantly, patients received daily doses of GM-CSF (75μg/d) for four days beginning two days before vaccination. After the first four cycles, vaccine was administered monthly. When patients showed signs of GvHD or infection vaccination was discontinued. WT-1-specific T cell responses were monitored in peripheral blood using tetramer analysis. No severe acute toxicity attributable to the vaccination was observed. In all patients a local inflammatory response at the site of injection was detected, which resolved fast after ending of the vaccination cycle. In some patients a systemic inflammatory response with fever and increase of the leukocyte count were observed, which also resolved a few days after vaccination. Three patients achieved a complete remission of the AML after transplantation and did not relapse. One patient developed an intraneural relapse of the AML in the N. medianus of the left arm, which was successfully treated by irradiation. Though, she did not show a relapse in the bone marrow and maintains a full donor chimerism in bone marrow and peripheral blood 600 days after transplantation. One patient relapsed and died four months after transplantation. Two patients developed a grade I GvHD of the skin shortly after the first vaccination. Therefore vaccination was discontinued and resumed two weeks after resolution of the GvHD. No GvHD signs were observed after the subsequent vaccination cycles. One patient developed a grade III GvHD of the skin and gut after the 3rd vaccination. In this patient GvHD proved to be resistant to the common immunosuppressive agents and the patients died of septicaemia four months after transplant. One patient developed a three-system grade IV GvHD after the 2nd vaccination. Vaccination was then resumed after resolution of the GvHD. He unfortunately died in complete remission because of systemic aspergillosis ten months after transplant. In three patients WT-1 specific T cell responses were monitored in peripheral blood at different time points prior and after vaccination by tetramer analysis. Prior to vaccination none of the patients showed a positive response to WT1. In all these patients CD8+/WT1-specific T cells were detected after one (1/3, 0,66% tetramer binding CD8+ cells) or two vaccinations (2/3, 0,99% and 0,81% tetramer binding CD8+ cells, respectively). In accordance with our observations, WT1 vaccination could contribute to the maintenance of a complete remission in patients with high-risk AML after HSCT. However, it could enhance GvH reactions because of the adjuvants used. Therefore further clinical observations in a larger number of patients are needed.

23 INVITED T regs in cancer

In the dog leukocyte antigen (DLA)-identical hematopoietic cell transplantation (HCT) model, stable marrow engraftment can be achieved with total-body irradiation (TBI) of 200 cGy when used in combination with postgrafting immunosuppression. The TBI dose can be reduced to 100 cGy without compromising engraftment rates if granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (G-PBMC) are infused with the marrow. T cell-depleting the G-PBMC product abrogates this effect. These results were interpreted to suggest that the additional T cells provided with G-PBMC facilitated engraftment by overcoming host resistance. We therefore hypothesized that the TBI dose may be further reduced to 50 cGy by augmenting immunosupression either by (1) tolerizing or killing recipient T cells, or (2) enhancing the graft-versus-host (GVH) activity of donor T cells. To test the first hypothesis, recipient T cells were activated before HCT by repetitive donor-specific PBMC infusions followed by administration of methotrexate (MTX) (n = 5), CTLA4-Ig (n = 4), denileukin diftitox (Ontak; n = 4), CTLA4-Ig + MTX (n = 8), or 5c8 antibody (anti-CD154) + MTX (n = 3). To test the second hypothesis, recipient dendritic cells were expanded in vivo by infusion of Flt3 ligand given either pre-HCT (n = 4) or pre- and post-HCT (n = 5) to augment GVH reactions. Although all dogs showed initial allogeneic engraftment, sustained engraftment was seen in only 6 of 42 dogs (14% of all dogs treated in 9 experimental groups). Hence, unless more innovative pharmacotherapy can be developed that more forcefully shifts the immunologic balance in favor of the donor, noncytotoxic immunosuppressive drug therapy as the sole component of HCT preparative regimens may not suffice to ensure sustained engraftment.

Unmanipulated HLA 2–3 Antigen-Mismatched (Haploidentical) Stem Cell Transplantation Using Nonmyeloablative Conditioning

The major problems in human leukocyte antigen (HLA)-mismatched stem cell transplantation (SCT) are graft failure and graft-versus-host disease (GVHD). Less-intensive regimens should be associated with a lower release of inflammatory cytokines and possibly less GVHD. The objective of this study was to investigate whether HLA-haploidentical SCT can be performed using nonmyeloablative conditioning and pharmacologic GVHD prophylaxis, including glucocorticoids. Using conditioning consisting of fludarabine, busulfan, and anti–T-lymphocyte globulin and GVHD prophylaxis consisting of tacrolimus and methylprednisolone (1 mg/kg/day), 26 patients who had hematologic malignancies in an advanced stage or with a poor prognosis underwent transplantation using peripheral blood stem cells from an HLA-haploidentical donor (2–3 antigen mismatches in the graft-versus-host [GVH] direction) without T-cell depletion. All patients except for 1 achieved donor-type engraftment. Rapid hematologic engraftment was achieved (neutrophils > 0.5 × 10 9/L on day 12 and platelets > 20 × 10 9/L on day 12), with full donor chimerism achieved by day 14. Fifteen patients did not develop acute GVHD clinically, and only 5 patients developed grade II GVHD. The recovery of CD4 + T cells was delayed compared with that of CD8 + T cells. Sixteen of the 26 patients are alive in complete remission. Four died of transplantation-related causes, and 6 died of progressive disease. These data suggest that nonmyeloablative conditioning, GVHD prophylaxis consisting of tacrolimus and methylprednisolone, and early therapeutic intervention for the GVH reaction allow stable engraftment and effectively suppress GVHD in HLA 2–3 antigen-mismatched SCT.

Graft‐versus‐host disease in recipients of grafts from natural killer T cell‐deficient (Jα281–/–) donors

Valpha14i natural killer T cells (NKT cells) are a CD1-restricted subset of NKT cells that express an invariable Valpha14+ Jalpha281+ alphabeta T-cell receptor. To determine whether the absence of Valpha14i NKT cells from the graft affects the development of acute GVHD, we induced GVH reactions using Jalpha281(-/-) mice as donors in the C57BL/6-->(C57BL/6 x DBA/2)F1-hybrid strain combination. Recipients of grafts from Jalpha281(-/-) donors were not protected from either the morbidity or the severe wasting syndrome associated with the development of acute GVHD, but the concentrations of some T helper 1 (Th1) and Th2 cytokines were different from those seen in recipients of grafts from wild-type donors. Interferon-gamma was seen earlier (day 4) in recipients of grafts from Jalpha281(-/-) donors but did not reach the concentrations seen in recipients of grafts from wild-type donors on day 8 (P < 0.02). On day 8, the amount of tumour necrosis factor-alpha released into the serum following the injection of a small amount of lipopolysaccharide was lower in recipients of grafts from Jalpha281(-/-) donors (P < 0.02). The amount of interleukin (IL)-5 was also lower in recipients of grafts from Jalpha281(-/-) donors, when compared to the concentration seen in recipients of grafts from wild-type donors (P < 0.002). IL-13 was seen in recipients of grafts from Jalpha281(-/-) donors but not in recipients of grafts from wild-type donors. Our findings show that the absence of donor Valpha14i NKT cells is associated with lower concentrations of some Th1 cytokines. We also observed higher IL-13 concentrations and lower IL-5 concentrations in recipients of grafts from Jalpha281(-/-) donors indicating a variable effect on Th2 cytokine production.

Single-Cell Cloning of Human, Donor-Derived Antileukemia T-Cell Lines for In vitro Separation of Graft-versus-Leukemia Effect from Graft-versus-Host Reaction

In previous studies, we showed the possibility of expanding in vitro polyclonal CTL lines directed against patient leukemia cells using effector cells derived from both HLA-matched and HLA-mismatched hematopoietic stem cell donors. Some CTL lines, especially those derived from an HLA-disparate donor, displayed residual alloreactivity against patient nonmalignant cells. In this study, we evaluated the possibility of separating in vitro CTLs with selective graft-versus-leukemia (GVL) activity from those potentially involved in the development of graft-versus-host disease (GVHD) through single T-cell cloning of antileukemia polyclonal CTL lines. We showed that CTLs that were expanded from a single T-cell clone (TCC), able to selectively kill leukemia blasts and devoid of alloreactivity towards nonmalignant cells, can be obtained from antileukemia alloreactive polyclonal CTL lines. TCCs expressed a wide repertoire of different T-cell receptor (TCR)-Vbeta families, mainly produced IFNgamma and interleukin 2, irrespective of CD8 or CD4 phenotype, and could be extensively expanded in vitro without losing their peculiar functional features. The feasibility of our approach for in vitro separation of GVL from GVH reaction opens perspectives for using TCCs, which are selectively reactive towards leukemia blasts, for antileukemia adoptive immune therapy approaches after hematopoietic stem cell transplantation, in particular from HLA-mismatched donors.

Immune restoration and/or augmentation of local graft versus host reaction by traditional Chinese medicinal herbs.

The in vitro restorative effect of aqueous extracts from two traditional Chinese medicinal herbs were studied in 19 cancer patients and in 15 normal healthy donors. Using the local graft versus host (GVH) reaction as a test assay for T-cell function, the extract from astragalus membranaceus (10 microgram/ml) induced a restored reaction in nine of ten patients with an increase in local GVH reaction from 18.2 plus/minus 15.8 mm3 to 112.9 plus/minus 94.2 mm3 (P less than 0.01). The extract from ligustrum lucidum, likewise effected an immune restoration in nine of 13 cancer patients with an increase in local GVH reaction from 32.3 plus/minus 36.1 mm3 to 118 plus/minus 104.9 mm3 (P less than 0.01). This degree of immune restoration appears to be complete as it equals the local GVH reaction observed among untreated mononuclear cells from normal healthy donors (82.8 plus/minus 41.1 mm3, P greater than 0.1). These results suggest that both extracts of the traditional Chinese medicinal herbs contain potent immune stimulants which may provide the rational basis for their therapeutic use as biological response modifiers.

Graft-versus-Leukemia and Graft-versus-Host Reactions after Donor Lymphocyte Infusion Are Initiated by Host-Type Antigen-Presenting Cells and Regulated by Regulatory T Cells in Early and Long-Term Chimeras

Regulatory T (T reg) cells and host antigen-presenting cells (APCs) have been implicated in graft-versus-host disease (GVHD) and the graft-versus-leukemia (GVL) effect after donor lymphocyte infusion (DLI), but their relative contributions remain unclear in early versus long-term complete donor or mixed murine allogeneic hematopoietic stem cell (HSC) chimeras. We have previously demonstrated that donor HSC-derived Thy1 + T reg cells, consisting primarily of CD4 +CD25 + cells, play an important role in the suppression of graft-versus-host (GVH) reactivity when DLI is given to complete donor chimeras 28 days after HSC transplantation. Data presented here demonstrate that protection against GVHD exerted by Thy1 + T reg cells is less evident with time and eventually is not required in long-term complete donor chimeras because of an absence of host-type APCs to activate alloreactive T cells. Lethal GVHD was observed when Thy1 + T reg cells were depleted from complete donor chimeras given by DLI at day 28, 35, or 42; however, T reg cell depletion and DLI at day 70 no longer induced GVHD-associated mortality. Moreover, the failure of DLI to induce GVHD with T reg depletion correlated with a loss of the DLI-induced GVL effect in long-term (day 100) complete donor chimeras. In contrast to the results from complete donor chimeras, GVL reactivity in day 100 mixed chimeras was robust after DLI. Loss of a DLI-induced GVL effect in long-term complete donor chimeras was attributed to the absence of host APCs because the infusion of exogenous host-type dendritic cells partially restored both DLI-induced GVL and GVH reactions in day 100 complete donor chimeras. The GVL and GVH reactions restored by infusion of host dendritic cells in day 100 complete donor chimeras were at least partially regulated by T reg cells because transient depletion of CD25 + cells increased both the GVL effect and the severity of GVHD after DLI. Taken together, these data suggest that T reg cells can regulate DLI-induced GVL and GVH reactions in both early and long-term complete donor chimeras, and a state of mixed chimerism is superior to complete donor chimerism because host-type APCs facilitate a DLI-induced GVL effect without severe GVHD.

Mucosal FOXP3+ regulatory T cells are numerically deficient in acute and chronic GvHD

CD4+CD25+ regulatory T cells (Tregs) control immune responses to self- and foreign antigens and play a pivotal role in autoimmune diseases, infectious and noninfectious inflammation, and graft rejection. Since recent experimental studies have indicated that Tregs were able to ameliorate graft-versus-host disease (GvHD), we analyzed the number of infiltrating Tregs in the intestinal mucosa as one site of GvH reactivity using immunoenzymatic labeling to enumerate FOXP3+ T cells in 95 intestinal biopsies from 49 allografted patients in comparison with healthy controls and patients with infectious inflammation. While patients with cytomegalovirus (CMV)-colitis or diverticulitis showed a concomitant increase of CD8+ effectors and Tregs, acute and chronic GvHD were characterized by the complete lack of a counter-regulation indicated by a FOXP3+/CD8+ T-cell ratio identical to healthy controls. In contrast, specimens without histologic signs of GvHD demonstrated increased numbers of FOXP3+ per CD8+ T cells, indicating that the potential for Treg expansion is principally maintained in allografted patients. Our findings provide evidence that GvHD is associated with an insufficient up-regulation of Tregs in intestinal GvHD lesions. The determination of FOXP3+/CD8+ ratio can be a helpful tool to discriminate GvHD from infectious inflammation after allogeneic stem cell transplantation.

Host Langerhans cells (LCs) can be therapeutically manipulated In vivo with imiquimod (TLR7 agonist) to augment DLI-mediated GVH and GVL reactivity

Host Langerhans cells (LCs) can be therapeutically manipulated In vivo with imiquimod (TLR7 agonist) to augment DLI-mediated GVH and GVL reactivity

Skin Explant Model of Human Graft-versus-Host Disease: Prediction of Clinical Outcome and Correlation with Biological Risk Factors

A human skin explant model has been used to predict the clinical outcome and to study the immunopathology of human graft-versus-host disease (GVHD). Whether the model gives the same predictive effect for GVHD in different hematopoietic stem cell transplantation (HSCT) settings has not been assessed. It is also unknown whether the skin explant result reflects the known biological risk factors for clinical GVHD. In this study, the skin explant model was used to detect graft-versus-host reactions (GVHR) in vitro for 225 eligible patient/donor pairs. The predicted skin GVHR grade was correlated with the outcome of clinical GVHD, as well as HLA matching status, sex mismatches, and patient age. In sibling HSCT under either myeloablative or reduced-intensity conditioning, a significant correlation was observed between the predicted skin GVHR and clinical GVHD ( P < .001 and P = .033, respectively). In HSCT using unrelated donors, the involvement of T-cell depletion led to a sharp increase in false-positive GVHR results, and no correlation was observed between the predicted skin GVHR and clinical GVHD. The skin GVHR grade correlated significantly with the HLA matching status (HLA-matched sibling pairs, HLA-matched unrelated pairs, and HLA-unmatched unrelated pairs). Furthermore, HLA-matched sibling pairs with a female-to-male sex mismatch had a significantly higher overall skin GVHR grade and a higher ratio of high- versus low-grade skin GVHR than the sibling pairs with all other sex combinations. Patient age was not reflected in the skin explant result. In conclusion, the predictive value of the skin explant model for aGVHD varies depending on the clinical transplant protocols, such as the type of GVHD prophylaxis used. Nevertheless, the skin explant model remains a unique in vitro system that provides an in situ histopathologic readout for studying alloreactivity and human GVHD. The model has also the potential to aid the development of novel prophylaxis and treatment for GVHD.

Modulation of Graft-versus-Host Disease: Role of Regulatory T Lymphocytes

Graft-versus-host (GVH) disease (GVHD) continues to be a major life-threatening complication after allogeneic bone marrow transplantation. Considerable progress has been made elucidating the pathophysiology of acute GVHD. Mature donor T cells transferred along with the marrow graft directly recognize antigenic differences on antigen-presenting cells of the host. Once activated, donor antihost-specific T cells can mediate tissue destruction. Interestingly, the failure to clonally delete autoreactive T cells in the thymus can also lead to an autoimmune syndrome mimicking the pathology of GVHD. Negative selection in the thymus may be compromised either by damage to the thymic epithelium (because of a direct attack by donor antihost alloreactive T cells) or by the use of immunosuppressive drugs that inhibit clonal deletion. An important component underlying GVHD mediated by either alloreactive or autoreactive T cells is the absence of a competent peripheral regulatory system. Studies in animal model systems clearly indicate that regulatory T cells play a vital role in down-regulating GVHD and are critically important for the establishment of active dominant tolerance to both allo- and self-major histocompatibility complex antigens. Although multiple populations of cells appear to participate in this process, CD4 + regulatory T cells that innately express CD25 + appear to orchestrate the regulatory control of the immune response. Evidence for regulatory T cells in clinical bone marrow transplantation, however, remains rudimentary. The recent identification that CD4 +CD25 + regulatory T cells preferentially express the Foxp3 nuclear transcription factor and the development of molecular reagents to isolate antigen-specific T cells have provided unique opportunities to explore immunoregulatory mechanisms after clinical marrow transplantation. Recent studies in recipients of clinical bone marrow transplantation suggest that antigen-specific CD4 +CD25 +Foxp3 + T cells play a vital role in the regulatory control of GVH reactions mediated by both alloreactive and autoreactive lymphocytes. These regulatory T cells also appear to facilitate the establishment of donor antihost and donor antidonor (self) tolerance.

Graft versus host disease in small bowel transplantation

Quantities of organized lymphoid tissue in small bowel allografts may cause graft versus host disease (GVHD) following transplantation. This study examines the effect of graft mesenteric lymphadenectomy on development of GVHD following small bowel transplantation in rats. GVH reactivity was assessed by measuring the degree of graft cell emigration to the host. In the PVG to DA strain combination, graft mesenteric lymphadenectomy led to a significant reduction in graft cell colonization of host lymphoid tissues from 40-50 per cent to 25-35 per cent. Transplantation from PVG to (PVG x DA)F1 hybrids caused fatal GVHD within 21 days whereas when DA donors were used survival was over 30 days. When mesenteric lymphadenectomy was performed on PVG donors, host survival increased by only 3-4 days. Mesenteric lymphadenectomy in DA donors led to long-term recipient survival with no GVHD. Intensity of GVHD following rat small bowel transplantation is a strain-dependent phenomenon and graft mesenteric lymphadenectomy does not always prevent GVHD. The mucosa may have an important immunological role.