GVHD In Mice Research Articles

Identification of a Major Susceptibility Locus for Lethal Graft-versus-Host Disease in MHC-Matched Mice

Graft-vs-host disease (GVHD) is the major cause of morbidity and mortality after allogeneic hemopoietic cell transplantation. From a genetic perspective, GVHD is a complex phenotypic trait. Although it is understood that susceptibility results from interacting polymorphisms of genes encoding histocompatibility Ags and immune regulatory molecules, a detailed and integrative understanding of the genetic background underlying GVHD remains lacking. To gain insight regarding these issues, we performed a forward genetic study. A MHC-matched mouse model was used in which irradiated recipient BALB.K and B10.BR mice demonstrate differential susceptibility to lethal GHVD when transplanted using AKR/J donors. Assessment of GVHD in (B10.BR x BALB.K)F(1) mice revealed that susceptibility is a dominant trait and conferred by deleterious alleles from the BALB.K strain. To identify the alleles responsible for GVHD susceptibility, a genome-scanning approach was taken using (B10.BR x BALB.K)F(1) x B10.BR backcross mice as recipients. A major susceptibility locus, termed the Gvh1 locus, was identified on chromosome 16 using linkage analysis (logarithm of the odds, 9.1). A second locus was found on chromosome 13, named Gvh2, which had additive but protective effects. Further identification of Gvh genes by positional cloning may yield new insight into genetic control mechanisms regulating GVHD and potentially reveal novel approaches for effective GVHD therapy.

Inflammatory Cytokine Induced Immunosuppression in Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are strongly immunosuppressive in vivo and in vitro in both animals and humans. Using our recently generated clones of MSCs derived from mouse bone marrow, we have shown that MSCs potently inhibit the T cell receptor (TCR)‐triggered proliferation and cytokine production by freshly‐isolated T cells. However, the molecular mechanisms controlling the immunosuppressive capacity of MSCs are unknown. We found that the immunosuppressive function of MSCs is elicited by IFNγ and the concomitant presence of any of three other proinflammatory cytokines, TNFα, IL‐1α or IL‐1β. These cytokine combinations provoke the expression of high levels of several chemokines and inducible nitric oxide synthase (iNOS) by MSCs. Chemokines drive T cell migration into proximity with MSCs, where T cell responsiveness is suppressed by nitric oxide (NO). This cytokine‐induced immunosuppression was absent in MSCs derived from iNOS−/− or IFNγR1−/− mice. Blockade of chemokine receptors also abolished the immunosuppression. Wild‐type MSCs, but not IFNγR1−/− or iNOS−/− MSCs, prevented GVHD in mice, an effect reversed by anti‐IFNγ or iNOS inhibitors. Wild‐type MSCs also inhibited delayed‐type hypersensitivity, while iNOS−/− MSCs aggravated it. Therefore, proinflammatory cytokines are required to induce immunosuppression by MSCs through the concerted action of chemokines and NO. We believe that a better understanding of the mechanisms through inflammatory cytokine activated‐MSCs mediate immunosuppression will lead to better clinical protocols for immune modulation.

Selective Depletion of CD11b+ Dendritic Cells from Murine Allogeneic Bone Marrow Grafts Promotes a Th1 Phenotype in Donor T Cells

Background: Allogeneic hematopoietic stem cell (HSC) transplant is a treatment used to cure patients with high risk and relapsed acute leukemia, via a graft versus leukemia (GvL) effect. In our previous studies, depletion of CD11b+ cells (containing CD11b+ dendritic cells, CD11b+ NK cells, and myeloid suppressor progenitor cells) from donor BM improved immune reconstitution and enhanced GvL effects without increased rates of GvHD in mice (Li, et al. BBMT 2004). In order to elucidate which subset of donor CD11b+ cells was responsible for this effect, we have transplanted combinations of FACS purified HSC, donor T cells, and CD11b- dendritic cells (DC) and found similar improvement in GvL activity associated with donor T cells polarized towards a Th1 phenotype (Li, et al. Blood 2007). In contrast, grafts containing FACS purified HSC, donor spleen T cells, and CD11b+ DC did not have significant GvL activity and donor T cells were polarized towards a Th2 phenotype. The objective of this study was to determine if selective depletion of CD11b+ dendritic cells by FACS would be a clinically relevant method, producing similar results as engraftment with purified populations of HSC, Cd11b- DC, and T cells.Methods: Selective CD11b+ DC depletion was achieved by FACS sorting in which cells from BM were sorted following gating of all nucleated cells with the exception of the CD11b+CD11c+Lineage- cells. The CD11b+ cells comprised ~1% of the BM. Undepleted BM was also stained and sorted using only a light scatter gate as a control for stress encountered during sorting. To study the long term effects of specific CD11b+ DC depletion, B10.BR or BA.B10 recipients were transplanted with 5×10^6 CD11b+ DC FACS-depleted or undepleted BM cells and 1×10^6 spleen T cells from C57BL/6J, Balb/C, and BA donors. Recipient mice were irradiated with 11Gy one day prior to the transplant. Recipient mice were monitored for survival, weight change, and GvHD score (based on weight change, activity, posture, fur texture, and skin condition) throughout the duration of the experiment and for chimerism of engraftment at days 30, 60, and 100 post transplant. Donor T cells were recovered from transplant recipients on days 3 and 10 post transplant and were examined for their Th1/Th2 polarization by flow cytometry after intracellular cytokine staining and ELISA of supernatants following short term culture.Results: B10.BR mice receiving undepleted BM from C57BL/6J or Balb/C mice had 100 day survivals of 60% and 40% respectively. Mice receiving CD11b+ DC depleted BM from C57BL/6J or Balb/C mice had equivalent survival at 80% and 60% survival, respectively, compared with mice receiving undepleted BM (p=ns). T cell chimerism at day 100 was over 95% donor chimerism for all mice, regardless of CD11b+ DC depletion. Differences in concentration of donor T cells in the blood were not significant between CD11b+ DC depleted and undepleted groups. The level of GvHD in recipient mice also did not differ significantly between groups. At day 3 post transplant, intracellular cytokine staining of donor T cells from mice receiving CD11b+ DC depleted BM had significantly higher levels of TNF-alpha compared with donor T cells from recipients of undepleted BM (p<0.05). Levels of IL-4, IL-10, IL-5 and IFN-gamma among donor spleen T cell population were not significantly different between undepleted and CD11b+ depleted BM recipients.Conclusions: Selective depletion of CD11b+ DC by FACS may be a clinically relevant method for increasing GvL activity without increasing GvHD. Depletion of CD11b+ DC from BM grafts polarizes donor T cells towards a Th1 phenotype, without increasing GvHD or affecting survival in recipient mice as compared with mice receiving undepleted BM grafts. The GvL effect of selective CD11b+ FACS depletion will be discussed.

Pathogenetic role of tissue factor in graft-versus-host disease

AbstractGraft-versus-host disease (GVHD) is a serious complication after allogeneic stem cell transplantation, the mechanism of it is still not elucidated. Recent findings suggest that host endothelial cells are a target of alloreactive donor cytotoxic T lymphocytes in GVHD and tissue factor (TF) plays an important role not only in coagulation-inflammation cycle, but also in transplant immunology. We postulate TF expression in vascular endothelial cells(VEC) may play an pivotal role in the pathogenesis of GVHD. TF gene andprotein expression in target organs of GVHD in aGVHD mice was significantly elevated compared to that of controls as determined by real-time PCR and Western blotting. Allogeneic CD4^+^T cell and CD8^+^T cells enhanced TF, VCAM-1, TNF-[alpha], IFN-[gamma] and IL-6 expression in TNF-[alpha] prestimulated HUVECs compared to controls as determined by flowcytometry and real-time PCR. JNK and p38MAPK mediated allogeneic T cells-induced TF expression in HUVECs. These effects were largely prevented by monoclonal antibody against TF, SB203580 and SP600125. In concert, these data provide strong evidence that upregulated TF expression is related to tissue damage caused by GVHD, TF isthe key factor in GVHD mediated by endothelial cells and allogeneic T cells-induced TF and consecutive proinflammatory cytokines expression in VEC contribute to the pathogenesis of GVHD.

Complement System Activation after Allogeneic Bone Marrow Transplantation May Early Predict the Development of Acute Gastrointestinal GVHD.

The Complement system consists of several serum proteins and receptors that belong to the innate immune response. It is activated by three possible pathways leading to the production of pro-inflammatory factors and the formation of the membrane attack complex. As it is known to be implicated in the physiopathology of several immune-mediated diseases, we investigated its role in GVHD in both humans and a mouse model. Thirty nine patients allografted for diverse haematological malignancies in our institution following myeloablative conditioning (n=22) or reduced intensity conditioning (RIC) (n=17) entered prospectively the study. Extensive exploration of the Complement system was carried out before and sequentially after allogeneic haematopoietic stem cell transplantation (HSCT). An activation of the classical pathway, as defined by a decrease of C3 and C4 proteins below normal values and of at least 50% of their pre-HSCT levels, was observed in 10/39 patients. Such activation was found in 9/22 (40%) patients who received myeloablative conditioning and in 1/17 (5.8%) patients allografted with RIC. This phenomenon occurred during the 4 first weeks after HSCT in 7 patients, at two months in one case and after withdrawal of immunosuppression in 2 patients. We observed a strong association between Complement activation and the development of acute gastrointestinal (GI) GVHD. Nine of the 10 patients who activated the Complement system (90%) developed GI GVHD within two weeks following the activation. By contrast only 4 of the 29 who did not activate the system, all of whom had received a RIC, developed GI GVHD (13.8%) (p<0.001). No correlation was found with the use of monoclonal antibodies, the degree of HLA and sex disparity between donor and recipient or the CMV status before the graft. We then analyzed Complement activation in a parent (C57BL/6, H–2b) to F1 [(C57BL/6×DBA2), H–bd] GVHD mouse model. Serum C3 Complement fraction and serum albumin protein (SAP) were measured sequentially after HSCT in lethally irradiated (9.75 Gy) mice reconstituted with either syngeneic or allogeneic bone marrow cells and splenocytes (107 and 13×106 cells/recipient, respectively). In comparison to naïve non HSCT recipient mice, the C3/SAP ratio was increased up to three times from Day 4 to 28 in syngeneic HSCT recipients (p=0.0002), reflecting the inflammatory response induced by the conditioning regimen without Complement activation in this conditioned non-GVHD control group. By contrast, the C3/SAP ratio was significantly decreased from Day 7 to 28 after HSCT in allogeneic recipients of HSCT in comparison to that of naïve (p=0.0005) and of syngeneic recipients (p=0.0002), indicating an activation of the Complement system in allografted mice that uniformly developed GI GVHD by Day 20 after HSCT. In conclusion, our results show that allogeneic HSCT induces Complement activation in both humans and mice, as a consequence of the immune alloresponse and not as a direct effect of the conditioning regimen. They also suggest that Complement C3 and C4 dosage might be useful in early prediction of acute gastrointestinal GVHD in humans. Ongoing studies are investigating the potential protective effect of pharmacological inhibitors of Complement activation on the development of GVHD in our mouse model.

Differential Regulation of Granzyme B and C Expression in Murine Cytotoxic T and NK Cells.

Cytotoxic lymphocytes, which include CD4+ and CD8+ T cells as well as NK cells, use the granule exocytosis pathway to kill virus-infected and tumor cells. Previous studies have characterized the expression of many genes in this pathway (e.g. perforin, granzyme A, granzyme B, etc.), although no reagent has been developed to measure granzyme C protein at the single-cell level. The murine granzyme C gene, orthologous to human granzyme H, lies 24.2Kb directly downstream from granzyme B in the granzyme B gene cluster. Recombinant granzyme C rapidly induces target cell death in a manner distinct from granzyme A- or B-induced death. To further understand the regulation and function of murine granzyme C, we developed a granzyme C-specific monoclonal antibody and used flow cytometry to examine the expression of granzyme C in resting and activated murine cytotoxic lymphocyte compartments. Naive CD4+ and CD8+ T cells express little or no granzyme C (0.1 + 0.1% of cells are positive). After activation of splenocytes with plate-bound CD3 and CD28 agonistic antibodies for 4 days, a small percentage of CD4+ (2.3 + 1.0% positive) and CD8+ (6.6 + 0.3% positive) T cells express granzyme C. However, only 24 hours later, almost all CD4+ (96.7 + 2.7%) and CD8+ (98.7 + 1.4%) T cells express granzyme C. Granzyme B co-staining revealed that granzyme B is detectable in both CD4+ and CD8+ T cells at least 48 hours before granzyme C is expressed. Furthermore, we employed a fully mismatched GVHD mouse model in order to examine T cell expression of granzymes B and C in vivo; similarly, granzyme B expression preceded granzyme C by at least 48 hours. In addition, CD4+Foxp3+ regulatory T cells also expressed granzyme C in this model. Murine NK cell granzyme C mRNA expression was assessed using Affymetrix microarrays (MOE430v2) at rest and following IL-15 activation. Resting NK cells express minimal granzyme C mRNA (mean gzmC probe set signal intensity + SD [n=3]): 356 + 202, which increased after IL-15 activation as follows: 4,180 + 1,106 (day 1), 60,788 + 8,455 (day 2), 167,448 + 26,398 (day 4), and 178,451+14,037 (day 6). Utilizing our granzyme C-specific mAb, we showed that very few resting murine NK cells express granzyme C protein (2.3 + 0.4% positive). Consistent with the mRNA data, the percentage of granzyme C-expressing NK cells was substantially increased after 3 days of IL-15 activation (88.8 + 5.2% positive). In contrast, granzyme B mRNA levels are high in resting NK cells (41,208 + 2,534), and increase only incrementally with IL-15 treatment (Fehniger et al., Immunity 2007 Jun;26(6):798–811). Granzyme B protein expression is controlled at a post-transcriptional level in NK cells, whereas granzyme C expression is transcriptionally regulated. Taken together, our findings show that the mouse granzyme B and C genes, despite being only 24.2Kb apart, are activated in cytotoxic lymphocytes with different kinetics; moreover, granzyme B and C protein abundance is controlled by completely different mechanisms in NK cells. These data suggest that the granzyme genes are differentially regulated in lymphocyte compartments, and that this regulation may be relevant for how cytotoxic lymphocytes function.

Expansion of Functional Polyclonal Human T-regulatory Cell Lines Using Artificial Antigen Presenting Cells and Rapamycin. (B159)

Abstract T-regulatory CD4+CD25+ T lymphocytes play a central role in regulating immune response and preventing autoimmunity. There is a broad spectrum of potential clinical applications of expanded T-regs including but not limited to treatments of graft versus host disease, transplant rejection, and type I diabetes. K562 cell line with stable expression of lentivirus-introduced CD64 and CD86 markers (KT64/86) and loaded with anti-CD3 antibodies was used to expand human CD4+CD25 high T lymphocytes. Though in several occasions stimulation of CD4+CD25high cells with KT64/86 only resulted in production of functional T-regulatory cells, only when antibiotic rapamycin was added to the cultures, functional Tregs were produced in 100% of stimulations. These cells were also active in vivo in reversing GVHD in NOG mice. Total CD4 lymphocytes grown in the presence of rapamycin though suppressive in vitro were not able to reverse GVHD in mice. Substitution of CD86 with CD80 resulted in a less potent Tregs. Addition of certain members of TNF receptor family such as CD70, OX40 ligand or 4-1BB ligand to KT64/86 cells, had different effects on expansion of Treg cell lines. While CD70 addition did not show any impact, 4-1BB and OX-40 ligands in most cases stimulated growth of T-regulatory cell lines but at the same time in most cases also attenuated their activity. The project is supported by JDRF Collaborative Center for Cell Therapy grant to C.J.

309: Blocking LFA-1 activation with lovastatin prevents graft-versus-host disease in mouse bone marrow transplantation

Leukocyte function associated antigen-1 (LFA-1) regulates T cell adhesion and activation. LFA-1 is constitutively expressed on cell surface in an inactive state. The control of LFA-1 activation is critical in inflammatory and immune responses. We demonstrated previously that the I-domain, the ligand binding site of LFA-1, changes from the low-affinity state to high-affinity state upon activation. Therapeutic antagonist, such as lovastatin, stabilizes the I-domain in the low-affinity state and inhibits the LFA-1 activation. Here, we report that lovastatin can block mouse T cell adhesion and proliferation in vitro. First, we demonstrated that lovastatin treatment reduced the mortality and morbidity in the mouse GVHD model. Lovastatin treatment significantly decreased GVHD mortality with 80% mice survived over 28 days, whereas more than 70% of the control mice died within the first 10 days, and the p values was 0.045. There were significantly reduced tissue damages in the skin, intestine and liver of lovastatin-treated mice. Second, we found lovastatin treatment reduced donor T cell homing to lymph nodes. There was a 65% reduction of CD4+ T cells homing to lymph nodes in lovastatin treatment group compared to control. The reduction of CD8+ T cells was greater with about 76% less cells homing to lymph nodes in the lovastatin treatment group. Third, we found lovastatin treatment reduced donor-derived T cell proliferation in vivo. There were 37% CD4+ and 31% CD8+ T cells remained undivided in the lymph nodes of the control mice at day 4 post-transplant. The lovastatin-treated mice had reduced proliferation kinetics of both CD4+ and CD8+ T cells with about 55% and 42% remained undivided. In the control lymph nodes, there were 42% CD4+ and 59% CD8+ T cells proliferated beyond 5th and 6th cell-divisions respectively, whereas the lovastatin treatment reduced the number to 31% and 48% respectively. In summary, we demonstrated here that lovastatin prevents both homing and proliferating of donor-derived T cells in the secondary lymphoid organs, which are crucial sites for alloreactive expansion. While most of the control mice died of acute GVHD within the first week of post-transplant when alloreactive T cells infiltrated the targeted organs, lovastatin treatment prevented the activation and expansion of donor-derived T cells, and thus reduced the GVHD mortality and morbidity. Our study provides rationale for a potential novel treatment for GVHD.

Allogeneic Cytokine-Induced Killer Cells Traffick to GVHD Target Tissue but Cause Less GVHD, While Retaining Tumor Cytotoxicity and Facilitating Bone Marrow Engraftment.

Cytokine-induced-killer cells (CIK), generated from splenocytes in mice and PBMC in human by the timed addition of IFN-γ, anti-CD3 MAbs and IL-2, express both T cell and NK cell markers and have cytotoxic activity against autologous and allogeneic tumors. We previously showed that adoptive transfer of allogeneic CIK in a murine model did not cause severe acute GVHD, leading to full survival. However the precise mechanism of reduced GVHD and retained GVT activity is not fully understood. To this end, we first evaluated the trafficking patterns of luciferase-transgenic CIK in a major MHC mismatched BMT model. In vivo bioluminescence imaging (BLI) showed that both CIK and fresh splenocytes homed to and proliferated initially in secondary lymphoid organs including the spleen, followed by infiltration of the gut and skin by day 7. All mice receiving fresh splenocytes died by day 10. Signal from gut mucosa in animals receiving allogeneic CIK cells was transient, however signals from other sites such as mesenteric lymph nodes were high and persistent. By day 21 allogeneic CIK reinfiltrated intestinal tissue without causing severe GVHD. To further investigate potential changes in the expression of surface molecules in vivo, we transplanted GFP positive fresh splenocytes and GFP positive CIK cells into major mismatched irradiated hosts, and then on day 7 analyzed activation markers, homing molecules, apoptosis related genes, and peripheral tolerance induction molecules such as CTLA-4 and PD-1 by FACS. No significant differences were noted between CIK and splenocytes. However we found that the cell division rate of CIK cells in vivo was much slower than those of splenocytes utilizing a CFSE based cell proliferation assay. This might lead to less GVHD in mice receiving CIK cells. Next we evaluated whether CIK retained tumor killing activity in vivo using A20 luciferase lymphoma cells into lethally irradiated MHC major mismatched receipients subcutaneously. We visualized the reduction of tumor by BLI. GFP positive CIK, which were extracted on day 7 following their transplantation into MHC-mismatched allogeneic recipients, retained tumor cytotoxicity in vitro. Finally, to study whether CIK facilitate BM engraftment, we quantified the absolute number of cells from the peripheral blood in MHC major-mismatched nonmyeloablative BMT. On day 30 mice receiving CIK showed rapid BM engraftment, especially in CD4 (p=0.0007) and CD8 (p<0.0001) subpopulations. In conclusion, allogeneic CIK homed to and expanded in GVHD target organs without causing acute GVHD, retained tumor killing activity and promoted BM engraftment.

Peyer patches are not required for acute graft-versus-host disease after myeloablative conditioning and murine allogeneic bone marrow transplantation

Graft-versus-host disease (GVHD) is a multistep disease process following allogeneic bone marrow transplantation (BMT). It has been postulated that the induction of acute GVHD requires the presence of Peyer patches (PPs). A new tumor necrosis factor (TNF)-deficient strain has been developed that totally lacks PPs and displays the defects characteristic of TNF ablation but not lymphotoxin-associated defects characterized by lack of both PPs and lymph nodes. To determine the necessity of PPs in acute lethal GVHD induction, we transplanted full major histocompatibility complex (MHC)-mismatched grafts into myeloablated TNF knockout recipients. No differences in the survival or GVHD-associated histopathologic lesions were observed between the recipients. We conclude that neither PPs nor host TNF-alpha is required for the development of acute lethal GVHD in mice that undergo myeloablative conditioning and allogeneic BMT.

Bone Marrow Derived, De Novo Generated CD4+ T Cells Are Previously Unsuspected Participants in CD8+ T Cell-Mediated Graft-Versus-Host Disease.

Although mature CD8+ T cells are known to be major effectors of acute GVHD, patients receiving T cell-depleted allografts remain at high risk for chronic GVHD. To what extent CD8+, CD4+ or both T cell subsets contribute to this chronic immunopathology is not known. We have recently demonstrated that alloreactive memory T cells develop in mice with acute GVHD and account for the persistence of host tissue injury (Journal of Immunology , 2005; 174:3051). Based on these findings, we now ask whether de novo generated donor T cells from engrafted T-BM themselves contribute to persistent host tissue injury in GVHD. Confirming previous observations, we found that transplantation of lethally irradiated C57BL/6SJL (B6, CD45.1) mice with highly purified C3H.SW (CD45.2) CD4+ naïve T cells did not cause GVHD, but mice receiving highly purified CD8+ naïve T cells together with C3H.SW T-BM, suffered severe acute GVHD. Surprisingly, in these mice receiving only CD8+ T cells, a substantial number of donor CD4+ T cells as well as CD8+ T cells were detected in GVHD target tissues, indicating that these infiltrating CD4+ T cells had arisen de novo from the transplanted T-BM. Donor CD4+ T cells recovered from GVHD mice expressed surface markers of activated effector/effector memory T cells, including CD25, CD69, CXCR3, and CD44hiCD62Llo. In response to host DCs, purified GVHD CD4+ T cells proliferated and expanded 4-5X more, and produced 10X higher levels of IFN-γ than did CD4+ T cells derived from B6 mice receiving C3H.SW T-BM alone. Furthermore, adoptive transfer of these in vivo generated GVHD CD4+ T cells, without CD8+ T cells, into secondary irradiated B6 recipients induced clinical GVHD characterized by delayed onset, weight loss, diarrhea, and lymphopenia, but without cutaneous inflammation. Histologic examination demonstrated chronic inflammation in the liver and intestinal tract, including epithelial apoptosis. Thymic pathology was dramatic in secondary B6 recipients of GVHD CD4+ T cells, including thymic atrophy, loss of thymic cortex, and infiltration of large amount of tingible macrophages. Taken together, these results demonstrate that donor bone marrow derived, de novo generated CD4+ T cells also contribute to GVHD together with transferred mature CD8+ T cells. Moreover, they suggest that these CD4+ T cells, in concert with alloreactive memory CD8+ T cells that develop during the evolution of GVHD, cause the persistence of acute GVHD and its subsequent progression into chronic GVHD. Thus, donor BM-derived, de novo generated CD4+ T cells are the “Hidden Dragon” of CD8+ T cell-mediated GVHD. Understanding how these CD4+ T cells are generated and regulated will prove to be critical to the prevention and treatment of both acute and chronic GVHD.

Prevention of graft-versus-host disease by a novel immunosuppressant, (5R)-5-hydroxytriptolide (LLDT-8), through expansion of regulatory T cells

(5R)-5-hydroxytriptolide (LLDT-8) is a new compound derived from triptolide, which is the major immunosuppressive fraction of Tripterygium wilfordii Hook. F (TWHF). In this study, we demonstrated that administration of LLDT-8 (1 mg/kg/day, p.o.) effectively prevented weight loss and death induced by allo-BMT (BLAB/c, H-2 d to C57BL/6, H-2 b), and extended survival in allo-BMT model of aGVHD. Following days 7 to 28 after allo-BMT, the allogeneic graft survived by increasing the number of engrafted cells (H-2 d) in spleens of recipient mice with LLDT-8 treatment. To construe the immunosuppressive effects of LLDT-8, the splenocytes (H-2 d) of LLDT-8 treated recipients (H-2 b) were tested for the proliferative responses to donor antigen (H-2 d), host antigen (H-2 b) and mitogen (ConA) stimulations, respectively, the results indicated that LLDT-8 induced the T cells' unresponsiveness to donor and host antigens, while still maintaining response to ConA; Compared with the vehicle group of GVHD mice, administration of LLDT-8 significantly inhibited T cells to produce IFN-γ with or without host antigen or ConA stimulation. Further studies indicated LLDT-8 had a normalizing effect on the ratio of CD4 + / CD8 + T cells, and increased CD4 +CD25 + T regulatory cells with the Foxp3 expression in splenocytes from LLDT-8 treated mice. The results outline the great potential of LLDT-8 as a therapeutic tool to induce suppression in GVHD.

Both apoptosis and complement membrane attack complex deposition are major features of murine acute graft-vs.-host disease

The parent-into-F1 mouse model (P→F1) of acute graft-vs.-host disease (GVHD) is a useful model of human acute GVHD because it allows the study of the T cell contribution to pathology without the complicating effects of conditioning regimens. To determine the similarity of this model to human GVHD, we assessed injury in organs typically involved in human acute GVHD (skin, liver) and less typically involved organs (spleen, kidney, lung). Mice were assessed histologically at early (2 weeks), intermediate (3 months) and late (6 month) time points. Based on the emerging roles of Fas ligand killing and complement deposition in allograft rejection, we correlated the amount of tissue specific TUNEL positive apoptosis and deposition of complement (C5b-9) with histopathologic changes. Our results indicate a striking similarity histologically between acute GVHD occurring in this model and in humans following bone marrow transplant. Moreover, C5b-9 deposition and apoptotic cell accumulation were found to parallel tissue injury in major organs of acute GVHD mice, although not all organs exhibited the same kinetic pattern. These results indicate a role for both adaptive immunity and innate immunity in this model of GVHD and support its use in modeling human acute GVHD in the nonmyeloablative setting.

Alleviation of chronic GVHD in mice by oral immuneregulation toward recipient pretransplant splenocytes does not jeopardize the graft versus leukemia effect

Chronic graft versus host disease (cGVHD) is the result of an immune-mediated attack by transplanted donor lymphocytes, entailing inflammatory damage to host target organs. Clinically, the post-bone marrow transplantation (BMT) graft versus leukemia (GVL) effect may be associated with GVHD. Immune hyporesponsiveness induced by oral antigen administration has recently been shown to prevent the development of cGVHD in a murine model. To evaluate whether amelioration of cGVHD in mice by induction of oral immune regulation in donors toward recipient pretransplant lymphocyte antigens is associated with attenuation of the GVL effect donor B10.D2 mice were fed with Balb/c splenocytes, B10.D2 splenocytes, bovine serum albumin (BSA), or regular chow, every other day for 10 days. Subsequently, transplantation of 2 × 10 7 splenocytes from donor B10.D2 mice to recipient Balb/c mice was undertaken, followed by inoculation of 3 × 10 3 BCL-1 leukemia on the day of BMT. Control groups were fed identically without leukemia inoculation. Mice were followed for survival and leukemia progression. Induction of tolerance was assessed by a mixed lymphocyte reaction (MLR). Cutaneous GVHD was assessed macroscopically. To elucidate the mechanism of any observed effect, serum interferon (IFN), interleukin (IL-2), IL-12, IL-4, and IL-10 were determined by enzyme-linked immunosorbent assay and flow cytometry analysis for CD4+, CD8+, and NK1.1+ lymphocyte subpopulations was performed. There was no significant difference in leukemia progression manifested by survival or white blood cell counts of orally immune-regulated mice compared with control animals. Cutaneous cGVHD was significantly ameliorated in Balb/c mice transplanted from tolerized B10.D2 mice. This effect was associated with a significant reduction in the mixed lymphocyte response of effector splenocytes from tolerized B10.D2 mice against Balb/c target splenocytes; significantly decreased serum IFN-γ and IL-2; increased serum IL-12 levels; increased peripheral NK1.1+ cells; and CD4+/CD8+ lymphocyte ratio. Oral tolerization of BMT donors toward recipient antigens ameliorates cGVHD without hampering the GVL effect.

The absence of Peyer’s patches or host TNF-A does not affect GVHD progression in an allogeneic BMT model using extensive conditioning regimens

Peyer’s patches (PP) have been previously shown to play a critical role in the initiation of acute lethal GVHD in a non-irradiated murine model (Murai et al., 2003). This report utilized IL-7 receptor antibody treatment of pregnant mice to inhibit the development of PPs in the fetus. We wanted to assess GVHD progression in mice congenitally deficient in PP presence and function. The absence of various members of the TNF and TNF-R families also affects secondary lymphoid structure and organization. The presence of PP has varied between different independently generated TNF knockout strains which may be related to the close linkages of tnf with lta and ltb genes. A new TNF deficient strain has been developed that lacks PPs, displays the defects characteristic of TNF ablation but not the LT associated defects characterized by lack of lymph nodes and defects in splenic microarchitecture. To examine the role of host-derived TNF family members in acute lethal GVHD, we also had to assess the role of secondary lymphoid structures. We utilized a full MHC mismatched mode of BALB/c (H2d) cells into myeloablated C57BL/6 (H2b) recipients. We lethally irradiated wild type (WT) mice, TNF-α deficient mice with Peyer’s patches (PP+) and TNF-α deficient mice without Peyer’s patches (PP-) followed by infusion of allogeneic bone marrow and spleen cells. We observed no difference in the survival of the three groups of mice (median day of death ranged from 26 to 27). In addition, no significant differences were observed in GVHD associated histopathological lesions in the small intestine. Hisopathological lesions observed in the colon were similar in both the PP+ and PP- TNF-α deficient recipients. Based on our observations, we conclude that (1) Peyer’s patches and (2) host TNF-α are not required for the development of acute lethal GVHD in mice that received extensive conditioning and allogeneic bone marrow transplantation.

Syngeneic reconstitution rescues mice that develop severe GVHD after allogeneic reconstitution

Background: Our Laboratory has been investigating donor specific hematopoietic reconstitution for the purpose of tolerance induction, a therapy currently limited by GVHD. This study investigates methods by which such GVHD can be managed. Methods and Results: We have established an acute GVHD mouse model by coinjection of T-cell depleted whole bone marrow (TCD-WBM) with CD4+ and CD8+-enriched splenocytes into lethally irradiated recipients. T cell depletion (TCD) was performed by extraction of CD3+, CD4+, CD8+, and NK1.1+ cells using magnetic beads. 25 Balb/c mice were given allogeneic 5 × 106 TCD-WBM plus 8 × 104 CD44/CD8+-enriched splenocytes. 5 mice were given 5 × 106 TCD-WBM alone. At day 9, 15 mice were rescued using 2, 5 and 10 × 106 syngeneic TCD-WBM, 5 mice in each group. GVHD was monitored by observing total body weight, body habitus, coat condition, and survival. At 42 days, survival of mice given TCD-WBM alone was 100% compared to 40% for mice TCD-WBM and CD4+/CD8+ splenocytes. Survival of mice given 2, 5, and 10 × 106 TCD-WBM was 60%, 60%, and 80% respectively. Weight data correlated as well. By day 16, the group which was given TCD-WBM alone was 10% below the start up weight compared to 41% for the group given TCD-WBM/splenocytes. Autologous rescue with 2, 5, and 10 × 106 TCD-WBM resulted in weights of 28. 28, and 22% of start-up weights, respectively. Peripheral blood of mice in all experimental groups was more than 98% donor-derived at 12 weeks. The group of mice given TCD-WBM/splenocytes and rescued with 2 × 106 autologous rescue cells did not survive beyond 9 weeks, but 99% of their peripheral blood cells was donor derived at 4 weeks. Conclusion: Such data demonstrate that acute GVHD can be alleviated without reverasal of donor-derived reconstitution using syngeneic rescue. These data suggest that such a method might prove useful for treatment of GVHD in methods of tolerance induction which utilize hematpoietic reconstitution.

Spontaneous activation of JNK-1 and PI-3 kinase can be induced in lupus-like chronic GVHD in the P-&gt;F1 model

Abnormalities in intracellular signaling pathways have been described in human SLE however, it is not clear whether they are primary, predisposing events or secondary to SLE expression. To address this question, we used a murine model of SLE (chronic P->F1 GVHD) and compared the function of a broad range of intracellular signaling pathways. In this model, SLE-like disease is induced in normal F1 mice by injection of parental CD4+ T cells and is mediated predominantly by Th2 cytokines. In contrast, acute GVHD is a cell-mediated anti-host response induced by CD4 and CD8 donor T cells and both Th1 and Th2 cytokines. Spleen cell lysates from control and GVHD mice (day 10 and 21) were immunoprecipitated with antibodies to Raf-1, ERK-1, JNK-1, p38 MAPK and PI-3 kinase and the function of the precipitated kinases determined using a specific substrate for each kinase. Raf-1 and ERK1 were selectively increased in acute GVHD (2-fold) only, whereas JNK-1 and PI-3 kinases were increased in both acute and chronic GVHD. This increase was approximately two fold greater for acute vs. chronic GVHD mice for both kinases. p38 MAPK was not increased over control in either form of GVHD and may reflect the earlier peak of IL-2 (day 3) in this model. The data suggest that Raf-1 and ERK-1 may be important in CD8 driven cell mediated immune responses whereas JNK-1 and PI-3 kinase signaling may be important in CD4 driven antibody mediated immune response. Studies are under way using purified T cell subsets to confirm this hypothesis. However these data support the idea that the signaling abnormalities reported in human SLE may be secondary to a continuous immune activation rather than indicative of a primary predisposing event.

Graft-facilitating doses of ex vivo activated gammadelta T cells do not cause lethal murine graft-vs.-host disease.

The purpose of this study was to examine the ability of gamma(delta) T cells to cause graft-vs.-host disease (GVHD) after allogeneic bone marrow transplantation (BMT) and to determine whether these cells offered any therapeutic advantages relative to alphabeta T cells. Due to the paucity of naive gamma(delta) T cells in mice and humans, gamma(delta), T cells (obtained from alpha(beta) T cell-deficient murine donors) were ex vivo activated and expanded in interleukin (IL)-2 so as to achieve sufficient cell numbers and to serve as a more clinically feasible strategy. After transplantation into lethally irradiated hosts, donor gamma(delta) T cells were detected in target organs of GVHD such as the spleen and intestines 2 weeks after BMT and constituted the primary T cell subpopulation. Large doses (150 x 10(6)) of activated gamma(delta) T cells, which we have previously shown capable of facilitating engraftment in MHC-disparate recipients, failed to cause fatal GVHD in lethally irradiated recipients of MHC-incompatible donor marrow grafts (C57BL/6 [H-2b]-->B10.BR [H-2k] and C57BL/6 [H-2b]-B6D2F1[H-2b/d]). The absence of GVHD was confirmed by histologic analysis of target organs, splenic B cell reconstitution, and appropriate negative selection in the thymus, that were all comparable to those observed in mice transplanted with T cell-depleted BM only. While early splenic reconstitution was attributable to donor gamma(delta) T cells, analysis of durably engrafted chimeras 2 months posttransplant revealed that the vast majority of donor splenic T cells expressed the alpha(beta) T cell receptor. The results of secondary adoptive transfer assays showed that these cells were tolerant of recipient alloantigens in vivo, demonstrating that gamma(delta) T cells did not prevent the subsequent development of donor anti-host tolerance in BM-derived alpha(beta) T cells. When comparatively evaluated, the minimal number of naive alpha(beta) T cells necessary for donor engraftment caused significantly more fatal GVHD than the corresponding minimal dose of activated gamma(delta) T cells and thus had a superior therapeutic index. These studies indicate that doses of activated gamma(delta) T cells that are able to promote alloengraftment do not cause lethal GVHD in mice transplanted with MHC-incompatible marrow grafts.

094 Possible involvement of dendritic epidermal T cells in the resistance to cutaneous GVHD in euthymic mice

094 Possible involvement of dendritic epidermal T cells in the resistance to cutaneous GVHD in euthymic mice

Enhanced graft-versus-host disease in older recipient mice following allogeneic bone marrow transplantation.

The incidence and severity of GVHD following bone marrow transplantation increases with recipient age. The role of recipient age on the development of GVHD was analyzed in a semi-allogeneic (C57BL/6-->(C57BL/6 x DBA/2)F1) murine GVHD system. Young adult (2 months) and old (12-14 months) recipient mice were lethally irradiated and reconstituted with young adult T cell-depleted bone marrow (ATBM) or ATBM and young spleen cells. A significantly higher percentage of old vs young recipients developed lethal GVHD. Furthermore, while pre-transplant conditioning with irradiation was not required to observe increased mortality in old recipients, irradiation predisposed the older animals for a more severe course of GVHD, suggesting that GVHD occurred in old compared to young animals in the absence of pre-transplant conditioning but was exacerbated by irradiation. Histologically, the immunological responses in the GVHD target organs were more severe in the old GVHD animals. In support of this observation, increased spontaneous proliferation was observed using lymphoid cells isolated from old vs young GVHD mice. These findings demonstrate that old recipients develop a more severe course of GVHD following BMT, and may present a unique opportunity to study age-related factors in the generation of GVHD.