Graft-versus-host Disease Controls Research Articles

Immune Profiling To Predict Treatment Response From Extracorporeal Photopheresis In Graft-Versus-Host Disease

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in patients receiving stem cell transplant. Extracorporeal photopheresis has demonstrated efficacy in a proportion of GVHD patients that are steroid refractory. However, given the expense, the challenging logistics, and unpredictable GVHD response to ECP, a predictive immune biomarker to assist with the selection of patients to treat with ECP can make this treatment modality more efficacious and cost-effective. One of the challenges of using leukocyte as biomarkers is that any single leukocyte population does not adequately describe the immune system. We have recently described an approach to characterize the immune system in a comprehensive manner. We have used a combination of whole blood quantitative flow cytometry and bioinformatics to generate immune profiles among patients with GVHD who were prescribed ECP. GVHD grading and clinical responses were confirmed by serial physical exams and review of medical records over a 6 month period. Response to ECP was defined as a decrease in clinical grade of GVHD (IBMTR and NIH consensus criteria) at the end of treatment. Peripheral blood drawn prior to initiation of ECP was used to generate a patient's immune phenotype by analyzing 33 lymphoid and myeloid surface marker combinations by flow cytometry. Immune phenotypes from 27 age-matched healthy volunteers were used as controls.From July 2011 to April 2013, 20 patients were enrolled (3 aGVHD, 17 cGVHD). At the time of this abstract, 15 patients were evaluable for treatment response, with 6 responders and 9 non-responders (median time on treatment 5.28 months, range 0.25 – 6 months). Using multivariate analysis by principal component analysis and unsupervised hierarchical clustering, we identified clustering patterns (immune profiles) in the composite immune phenotypes of the GVHD patients and controls. The healthy controls clustered together into a single immune profile. Four of the six responders clustered closely together and away from the 9 non-responders and healthy controls. Anova analysis identified 21 individual markers that were different among the clusters. Using non-parametric Wilcoxon comparison, we found that patients who responded to ECP had lower numbers of lymphocytes (p=2.5x10-12), CD4 T cells (p=0.002), CD4+CD69+ T cells (p=0.02), and CD8+CTLA4+ (p=1.8x10-5) than healthy controls and non-responders.To our knowledge, this is the first report of a bioinformatics approach to comprehensively examine systemic immune profiles of GVHD patients. Our results provide the proof-of-concept for using this method to identify an immune profile as a predictive biomarker in the treatment of GVHD. Our data supports the need for a larger study to definitively define the immune profiles and validate this approach. Disclosures:No relevant conflicts of interest to declare.

Connective Tissue Growth Factor Is Overexpressed in Explant Lung Tissue and Broncho-Alveolar Lavage in Transplant-Related Pulmonary Fibrosis.

BackgroundConnective tissue growth factor (CTGF) is an important mediator in several fibrotic diseases, including lung fibrosis. We investigated CTGF-expression in chronic lung allograft dysfunction (CLAD) and pulmonary graft-versus-host disease (GVHD).Materials and MethodsCTGF expression was assessed by quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry in end-stage CLAD explant lung tissue (bronchiolitis obliterans syndrome (BOS), n=20; restrictive allograft syndrome (RAS), n=20), pulmonary GHVD (n=9). Unused donor lungs served as control group (n=20). Next, 60 matched lung transplant recipients (BOS, n=20; RAS, n=20; stable lung transplant recipients, n=20) were included for analysis of CTGF protein levels in plasma and broncho-alveolar lavage (BAL) fluid at 3 months post-transplant, 1 year post-transplant, at CLAD diagnosis or 2 years post-transplant in stable patients.ResultsqPCR revealed an overall significant difference in the relative content of CTGF mRNA in BOS, RAS and pulmonary GVHD vs. controls (p=0.014). Immunohistochemistry showed a significant higher percentage and intensity of CTGF-positive respiratory epithelial cells in BOS, RAS and pulmonary GVHD patients vs. controls (p<0.0001). BAL CTGF protein levels were significantly higher at 3 months post-transplant in future RAS vs. stable or BOS (p=0.028). At CLAD diagnosis, BAL protein content was significantly increased in RAS patients vs. stable (p=0.0007) and BOS patients (p=0.042). CTGF plasma values were similar in BOS, RAS, and stable patients (p=0.74).ConclusionsLung CTGF-expression is increased in end-stage CLAD and pulmonary GVHD; and higher CTGF-levels are present in BAL of RAS patients at CLAD diagnosis. Our results suggest a potential role for CTGF in CLAD, especially RAS, and pulmonary GVHD.

α4β7 Blockade with Vedolizumab Is Effective in Prevention and Treatment of Experimental Acute Graft Versus Host Disease

Introduction Graft versus host disease (GVHD) leads to significant post-transplant morbidity and mortality after allogeneic hematopoietic cell transplant (allo-HCT). Vedolizumab (VDZ) is a monoclonal antibody targeting the homing of T cells to the intestine through binding of integrin α4β7 of their surface. We hypothesized that interfering with T cell homing to the GI tract results in decreased GVHD severity and improved outcome in an established humanized mouse model of allo HCT. Methods Sublethally irradiated RAG2-/- γc-/- mice were transplanted with 30 million human PBMCs (huPBMCs). Vedolizumab or control IgG was given intraperitoneally at a dose of 4mg/kg body weight on day -1 and day +15 (preventive approach) or day +8 and day +22 (therapeutic approach). Time points were chosen in order to start α4β7 blockade prior to clinical onset of GVHD (preventive approach) and when animals started to get significantly suffer from GVHD (therapeutic approach). As non-GVHD controls, animals received sublethal irradiation without huPBMCs administration and received VDZ or control IgG to assess for direct conditioning and VDZ toxicity. Results HuPBMC CD45 splenocyte analysis on day +28 after infusion demonstrated strong human leukocyte engraftment with more than 80% of spleen cells being huCD45 positive. Both preventive and therapeutic administration of Vedolizumab resulted in significantly improved survival in HuPBMC treated recipients when compared to IgG treated GVHD controls at week 8 (62.6% Vs 12.5% and 50% Vs 12.5% respectively). All recipients, which did not receive huPBMCs survived, indicating VDZ safety in this model. Clinical GVHD scores after day 21 after transplant were significantly lower in VDZ huPBMCs treated animals until end of analysis compared to control-treated recipients using both the preventive and therapeutic approaches, along with significant decreases in GI tract pathology scores on day +28. No differences in pathology were seen in liver or lung at this time point. Serum cytokine analysis revealed decreases in TNF (p Conclusion Vedolizumab reduces the severity of intestinal GVHD by targeting donor T cell migration into the intestinal tract both when given early prior to onset of clinical disease and given at later time point, and improves overall survival both in the preventive and therapeutic setting. Given these promising results, targeting T cell homing with VDZ potentially presents a novel option for the management of intestinal GVHD.

Graft Versus Host Disease (GvHD) Is Attenuated By Administration of Pregnancy Specific Glycoproteins through Induction of Immune Tolerance

Hematopoietic stem cell transplantation is curative for many disorders; however, it can be associated with significant morbidity and mortality, often as a result of graft versus host disease (GvHD). GvHD is an immune mediated reaction in which donor T-cells recognize the host antigens as foreign, causing donor T-cells to proliferate and attack host tissues. Establishment of a tolerogenic immune environment while preserving immune response to infectious agents and malignancy is required for successful bone marrow transplantationand GvHD presents a significant obstacle to this.Pregnancy specific glycoproteins (PSGs) are synthesized by the placenta at the onset of pregnancy and are believed to play a role in maintaining a tolerogenic immune environment to prevent rejection of the fetus by the maternal immune system. Two specific PSG proteins, PSG1 and PSG9, are of particular note as they have both been shown to be involved in pathways devoted to induction of immune tolerance. Both PSG1 and PSG9 are involved in activation of transforming growth factor-β (TGFβ), a cytokine essential to suppression of inflammatory T-cells and important for differentiation of tolerance inducing CD4+ CD25+ FoxP3+ regulatory T-cells (Tregs), a cell population shown to be important in the prevention of GvHD. We thus hypothesized that PSG1 and PSG9 (PSG1/9) could be used to treat or prevent GvHD by inducing immune tolerance through TGFβ.Using surface plasmon resonance (SPR) analysis, we have shown that PSG1/9 bind directly to the latency associated peptide (LAP), a protein that confers latency to mature TGFβ, effectively blocking binding of TGFβ to its receptors. Further, bioassays and ELISA data show an increase in the levels of bioactive TGFβ after treatment with PSG1/9, indicating that these proteins have a role in TGFβ activation through their interaction with LAP. In vitro data using naïve mouse T-cells showed that upon treatment with PSG1, there was a significant increase of CD4+ CD25+ cells expressing FoxP3 (18%±1.5; n=3) compared to only 2% in untreated controls (2%±0.4; n=6) (p<0.0001). Similarly, PSG9 treatment also increased FoxP3 expression (11%±2.8; n=3) over controls (p<0.0001). The increase in FoxP3 expression was also observed upon treatment of primary naïve human T-cells with both PSG1 and PSG9 (PSG1, p=0.0073; PSG9, p=0.0028). When a TGFβ receptor inhibitor was added to cell culture, the increase in FoxP3 expression was effectively blocked, further supporting the hypothesis that PSG1/9 induce expression of FoxP3+ Tregs through regulation of TGFβ. As IL-2 is important for the stability and differentiation of Tregsin vivo and its transcription is suppressed by TGFβ, we performed bioassays on CD4+ naïve T-cells with no added IL-2. We observed that in the absence of added IL-2, treatment with PSG1/9 resulted in an inhibition of IL-2 secretion by activated CD4+ T-cells, and there was no increase in the number and percentage of FoxP3 expressing cells.SBE-luc mice, which express luciferase in response to activation of the TGFβ-SMAD pathway, exhibited significantly increased luciferase expression in the abdomen when injected intraperitoneally with PSG1. This supports the idea that PSG1 induces expression of FoxP3 through the TGFβ receptor pathway in vivo. Finally, using a murine model of GvHD, we observed that mice receiving PSG1 had reduced numbers of infiltrating inflammatory CD3+ T-cells in the colon and showed a marked improvement physically and histologically over untreated controls. In addition, PSG1 treated mice had significantly higher expression of FoxP3 in CD4+ CD25+ splenic cells (30%±9.6; n=4) when compared to untreated GvHD controls (8.6%±3.2; n=3) (p=0.0287).Currently, we are studying the cytokine profiles of mice treated with PSG to determine the efficacy of PSG1/9 in reducing the release of pro-inflammatory cytokines during GvHD. Overall, our data suggests that PSG1/9 induce immune tolerance in GvHD and may be a future treatment option for these patients. DisclosuresNo relevant conflicts of interest to declare.

Early cell-cycle gene expression in T-cells after hematopoietic stem cell transplantation

Regeneration of the immune system after hematopoietic stem cell transplantation (HSCT) is a slow process. Early cell cycle proto-oncogenes are key players in the first events of the proliferative process of T-cell immune response. To identify the causes of the prolonged immuno-suppression after transplantation we evaluated the expression of early T-cell cycle genes c-myc, c-jun and c-fos in peripheral blood T-cells from post-transplant patients versus healthy controls before and after phytohemagglutinin (PHA) incubation. Results show that c-jun and c-fos expression continued to increase during the first 18months post-transplant, and eventually decreased at two years post-transplant. C-myc, c-jun and c-fos expression values also showed a time-dependent increase in surviving patients. In non-surviving patients, however, there was a time-dependent decrease in gene expression. Significant correlation was shown in c-jun expression values of all patients up to 2years post transplant between resting state and 3h post-PHA incubation when compared to healthy controls. A positive correlation was found in c-fos gene expression between values at resting state and 3h post PHA incubation in patients with Graft versus Host Disease (GVHD) compared patients with no GVHD or healthy controls. A positive correlation was observed between white blood cell count and c-fos expression among non-surviving patients and patients who suffered from GVHD.Our results show that molecular monitoring of the immune reconstitution using c-jun, and c-fos expression combined with clinical parameters may improve the early detection of immune-function failure. This follow-up system may allow for the development of more efficient treatments and new interventions designed to hasten desirable T-cell regeneration.

Next-Generation Sequencing for the Identification of Transplantation-Associated Pathogens

AbstractAbstract LBA-4 Background:Hematopoietic stem cell transplantation (HSCT), while a powerful tool in the clinician's armamentarium against refractory hematological diseases, is associated with high non-relapse mortality. Infectious, immunological and idiopathic syndromes all contribute to the nearly 30% treatment related mortality in the first year post-transplantation. Microbiological triggers have been suggested in many of the idiopathic post-transplantation syndromes that occur, but actual pathogens have been discovered in only a small subset.Recently, Herrera and colleagues described an antibiotic-responsive colitis unique to patients who have undergone umbilical cord stem cell transplantion.1This “cord colitis syndrome” (CCS) is clinically and histopathologically distinct from other known causes of colitis in transplantation patients, such as viral colitis, bacterial colitis and intestinal graft versus-host disease (GVHD). Contemporary microbiological tools did not reveal a causative organism in CCS. We therefore sought to apply an unbiased, “deep sequencing” approach for the discovery of a potential CCS-associated pathogen. Methods:Of the 11 patients described in the original CCS cohort, five patients had undergone colonic biopsies in the time frame just before and just after diagnosis with and treatment for CCS. We selected these formalin fixed, paraffin embedded biopsies, which measured approximately 2mm × 2mm × 1mm, and extracted DNA. Four samples (two temporally separated biopsies from two patients) had adequate DNA to perform whole genome sequencing (WGS). Following sequencing at two separate facilities (to control for potential sources of contamination), we performed taxonomic classification of all resultant sequencing “reads” using the PathSeq software.2Computational assembly of unclassifiable “reads” was performed for the discovery of novel microbial organisms. An attempt was then made to investigate for the presence of candidate microbes identified by this method in the three additional patients for whom colonic biopsies were available and in various controls. Results:Using this approach, a large proportion of non-human reads (over 2.5 million high-quality reads) from all four sequenced samples were unclassifiable, suggesting the presence of a yet-undiscovered microbial organism within the tissue specimens. In order to infer the genomic sequence of this organism, computational assembly was performed with reads from a single patient using two different DNA assembly programs (VELVET and AllPaths-LG).3,4 Both assembly methods independently resulted in the generation of a nearly 7.65 Mb draft genome. Ninety-eight of 99 contiguous overlapping sequences (“contigs”) demonstrated homology to Bradyrhizobium species. This novel organism was provisionally named Bradyrhizobium entericabased on the results of a rooted phylogenetic analysis. PCR confirmed the presence of B. enterica in three additional CCS patients and demonstrated absence of B. enterica in normal colon, colon cancer and GVHD controls. Conclusions:Using an unbiased, sequencing-based method for pathogen discovery, we report the discovery of Bradyrhizobium enterica, a novel bacterium, from colon biopsies of patients affected with CCS. This is the first demonstration, to our knowledge, of the discovery of a novel bacterium through sequencing of a diseased human tissue. We showed that B. enterica is present in additional cord colitis patients and is absent in normal colon, colon cancer and GVHD controls. Given the ease, rapidity and culture-independent method of discovery of a disease-associated bacterium that was demonstrated in this previously idiopathic syndrome, we anticipate that this methodology will be more widely applied in the field of stem cell transplantation for the identification of potential disease-associated pathogens. Disclosures:Marty:Chimerix: Consultancy, Research Funding. Meyerson:Foundation Medicine: Consultancy, Equity Ownership; Microgenomica: Equity Ownership; Novartis: Consultancy.

Basement membrane antibodies in sera of haematopoietic cell recipients are associated with graft‐versus‐host disease

Graft-versus-host disease (GvHD) occurs frequently after haematopoietic cell transplantation (HCT). Mucocutaneous lesions of GvHD may mimic bullous autoimmune dermatoses, and 10 cases of concurrent GvHD and a bullous autoimmune disease have been reported in the literature. To determine the frequency of circulating antibodies to the cutaneous basement membrane zone (BMZ) in HCT patients with GvHD in comparison with HCT patients without GvHD, psoriasis patients and healthy controls. We examined 42 patients with chronic GvHD, 18 HCT patients without GvHD, 11 psoriasis patients and 40 healthy controls, prospectively. Sera were tested by indirect immunofluorescence (IIF) on salt-split skin, NC16a-ELISA and immunoblot using keratinocyte extracts. Univariate statistical analyses and logistic regression were performed to assess possible correlations of graft and patient characteristics with the presence of BMZ antibodies. Circulating basement membrane zone (BMZ) antibodies were detected in 10/42 (24%) GvHD sera by immunoblot, but not in any of the HCT sera from patients without GvHD (0/18; 0%). The antibodies targeted collagen VII, BP230, collagen XVII/BP180 or p200/laminin gamma1. Clinically manifest bullous autoimmune dermatoses (bullous pemphigoid or epidermolysis bullosa acquisita) were found in two GvHD patients. 1/11 (9%) psoriasis sera and 1/40 (2.4%) healthy control sera reacted with collagen XVII or BP230, respectively. Circulating BMZ antibodies are significantly associated with chronic GvHD in contrast to uncomplicated HCT. Recurrent mucocutaneous lesions in chronic inflammatory skin disorders may liberate antigens, which may lead to production of BMZ antibodies, particularly in the context of GvHD-mediated reduced self-tolerance.

Cutaneous T cell lymphoma and graft-versus-host disease: A comparison of in vivo effects of extracorporeal photochemotherapy on Foxp3+ regulatory T cells

Extracorporeal photochemotherapy (ECP) is a well established treatment for both cutaneous T cell lymphoma (CTCL) and graft-versus-host disease (GVHD). However, the general effector mechanism is not fully settled. Twenty-four patients with CTCL and 14 patients with GVHD were included to assess the relative numbers of regulatory T cells (Treg) and any change in the serum cytokine profile during 6 months of ECP therapy. The relative amount of Treg cells was twice as high in CTCL compared to GVHD and healthy controls. TGF-beta was on average three times higher in GVHD than in CTCL. Both patient groups had a small but significant increase in TGF-beta after treatment. Our results indicate a strengthened Treg function as a result of ECP. Elevated TGF-beta may indicate high Treg activation in GVHD, whereas an increased number of Treg cells in CTCL could be interpreted as a response that is involved in down-regulating the lymphoma cells.

Th2 Cell Therapy of Established Acute Graft-Versus-Host Disease Requires IL-4 and IL-10 and Is Abrogated by IL-2 or Host-Type Antigen-Presenting Cells

Delayed donor Th2 cell infusion permits a graft-versus-tumor (GVT) effect to occur with subsequent amelioration of established graft-versus-host disease (GVHD). Relative to GVHD controls (B6-into-BALB/c model), recipients of delayed Th2 cells (day 14 post-BMT) had increased survival (3/3 experiments [exp]; each exp P < .0001) and reduced GVHD by histology analysis 5 days post-Th2 infusion without increased tumor burden (3 of 3 exp; each exp P ≤ .02). Th2 cell-mediated amelioration of GVHD was associated with greatly reduced allospecific IFN-γ secretion, in vivo augmentation of allospecific IL-4 and IL-10 secretion, and reduction in donor CD8+ T cell number post-BMT (3 of 3 exp; each comparison, P ≤ .003). To better understand the molecular mechanism of this GVHD therapy, Th2 cells were generated from wild-type (WT), IL-4 deficient (KO), or IL-10 KO donors: remarkably, recipients of IL-4 or IL-10 KO Th2 cells had no survival advantage, no improvement in GVHD by histology, no reduction in CD8+ T cell expansion post-BMT, and no in vivo shift toward type II cytokines. We reasoned that IL-2 and alloantigen availability may be limiting factors for Th2 cell therapy, and as such, evaluated whether coadministration of IL-2 or coinfusion of host-type antigen-presenting cells (APC) might intensify the anti-GVHD effect. However, contrary to these hypotheses, concomitant IL-2 therapy or APC administration fully abrogated the Th2 cell-mediated survival advantage and histology-defined GVHD reduction, reduced Th2 cell expansion in vivo while promoting CD8+ T cell expansion from cells originating from the initial allograft, and impaired type II polarization in vivo. In conclusion, Th2 cell therapy can rapidly ameliorate severe GVHD via IL-4 and IL-10 mediated mechanisms, and potentially, via IL-2 consumption and APC modulation mechanisms.

CCL8 is a potential molecular candidate for the diagnosis of graft-versus-host disease

AbstractAlthough graft-versus-host disease (GVHD) is a life-threatening complication of hematopoietic stem-cell transplantation (HSCT), its current diagnosis depends mainly on clinical manifestations and invasive biopsies. Specific biomarkers for GVHD would facilitate early and accurate recognition of this grave condition. Using proteomics, we screened for plasma proteins specific for GVHD in a mouse model. One peak with 8972-Da molecular mass (m/z) retained a discriminatory value in 2 diagnostic groups (GVHD and normal controls) with increased expression in the disease and decreased expression during cyclosporin A treatment, and was barely detectable in syngeneic transplantation. Purification and mass analysis identified this molecule as CCL8, a member of a large chemokine family. In human samples, the serum concentration of CCL8 correlated closely with GVHD severity. All non-GVHD samples contained less than 48 pg/mL (mean ± SE: 22.5 ± 5.5 pg/mL, range: 12.6-48.0 pg/mL, n = 7). In sharp contrast, CCL8 was highly up-regulated in GVHD sera ranging from 52.0 to 333.6 pg/mL (mean ± SE: 165.0 ± 39.8 pg/mL, n = 7). Strikingly, 2 patients with severe fatal GVHD had extremely high levels of CCL8 (333.6 and 290.4 pg/mL. CCL8 is a promising specific serum marker for the early and accurate diagnosis of GVHD.

Role of STAT1 Expression during Graft-Versus-Host Disease (GVHD) in the Gastrointestinal Tract: Association with Lamina Propria Cell Infiltration and Tissue Cytokine/Chemokine Expression.

The role of the IFN-g in the development of GVHD remains enigmatic. Whereas it has been shown that GVHD can occur in the absence of IFN-g, there are partially contradicting results how IFN-g can modulate GVHD. Thus, it has been suggested that blocking IFN-g might ameliorate gut GVHD in rodent BMT models and its direct cytopathic effects in human ex vivo skin explant models. However, there is also data supporting the notion that IFN-g is important for limiting GVHD by induction of activation induced cell death (AICD) in donor T cells. Furthermore, it has been demonstrated in preclinical BMT models that IFN-g may accelerate or mitigate GVHD depending on the conditioning intensity. We have recently demonstrated that mitigation of GVHD by treatment with the Histone deacetylase Inhibitor (HDACi) suberonylanilide hydroxamic acid (SAHA) is associated with early downregulation of STAT1 phosphorylation in the spleen and host epithelial GVHD target organs. To further understand the role of STAT1 in the development of GVHD we studied STAT1 and p-STAT1 (Tyr701) expression by immunohistochemistry in the GVHD target organs liver, small bowel and colon following induction of GVHD and correlated these findings with the presence of lamina propria (LP) lymphocytes, typical features of GVHD-induced tissue damage (crypt cell apoptosis, crypt regeneration) and expression of tissue cytokines/chemokines. GVHD was induced in the fully MHC mismatched BALB/c to B6 strain combination following lethal irradiation with 975 rad and animals were sacrificed on days +1, +3 and +6. As detected by western blots p-STAT1 expression became detectable on day +1 in the spleen and on days +3 in the liver, small bowel and colon. Compared to untreated controls immunohistochemical p-STAT1 staining became apparent on day +3 post-BMT in the small bowel and colon of syngeneic controls and GVHD animals. In syngeneic controls p-STAT1 expression decreased again on day +6. In contrast, a further significant increase in p-STAT1 staining was observed in animals with GVHD in the colon and small bowel on day +6. In the colon this significant increase in crypt cell p-STAT1 staining was associated with the presence of LP infiltrating lymphocytes and coincided with the maximal features of tissue damage (luminal sloughing, crypt destruction and crypt apoptosis). In line with these results IFN-g protein expression became detectable in colon tissue lysates on day +6 supporting the role of IFN-g producing infiltrating donor T cells in causing STAT1 activation and tissue damage. We conclude that in comparison to untreated controls STAT1 activation can be observed in the colon and small bowel starting on day +3 in animals with GVHD and syngeneic controls. Whereas pSTAT1 staining peaks at day +3 in syngeneic controls and declines thereafter, maximal STAT1 activation occurs in GVHD animals on day +6, coincides with detectable IFN-g expression and is accompanied by LP infiltration and features of severe GVHD-related tissue damage. To fully understand the role of IFN-g in the development of gut GVHD further studies are warranted to delineate the role of STAT1 dependent and independent signaling pathways in the development of GVHD.

Secondary Lymphoid Tissues Are Not Required for the Induction of GVHD Following Allogeneic BMT: A Shifting Paradigm for T Cell Allo-Activation.

Allogeneic (allo) bone marrow transplantation (BMT) is the only curative option for many patients with malignant and non-malignant diseases. Acute graft versus host disease (GVHD) is the major complication of allo-BMT and limits the utility of this treatment strategy. The induction of GVHD fundamentally depends upon the activation of donor T cells by host antigen presenting cells (APCs), and the prevailing hypothesis is that these critical interactions occur in secondary lymphoid organs, such as lymph nodes (LN) Peyer's patches (PP), and spleen (SP). We tested this hypothesis by using a well established, MHC disparate, murine SCT system (Balb/c → B6) and homozygous aly/aly (alymphoplasia) mice that are deficient in all LN and PP and heterozygous aly/+ littermate controls. Lethally irradiated, splenectomized, aly/aly mice (LN/PP/SP −/ −) and aly/+ sham mice (LN/PP/SP +/+) received BMT either from syngeneic (aly/aly) or allo (Balb/c) donors. In some experiments, wild-type B6 recipients of B6 or Balb/c BMT served as additional negative and positive GVHD controls respectively. The severity of GVHD was assessed by survival and well-described scoring systems of both clinical and target organ disease. As expected, greater than 95% of syngeneic (syn) BMT recipients survived and were indistinguishable from naïve, un-transplanted controls, whereas LN/PP/SP +/+ mice receiving allo-BMT showed significant signs of GVHD with ~40% mortality by day 49. All LN/PP/SP −/ − allo-BMT recipients also survived, but surprisingly, examination demonstrated that they too developed significant clinical GVHD compared to syn controls (score: 3.2 vs. 0.85) that was comparable in severity to LN/PP/SP +/+ mice (3.1). Moreover, histopathologic analysis demonstrated that LN/PP/SP −/ − allo-BMT recipients developed significantly greater GVHD target tissue damage in the liver, intestinal tract and skin compared to syn controls. In fact, LN/PP/SP −/ − allo-BMT recipients developed more severe hepatic GVHD compared to allo littermate (LN/PP/SP +/+) controls (30.8±1.9 vs. 20.7±2.2; p < 0.01). Similar differences in liver GVHD was also seen between allo groups as early as day 7 (16.0±2.2 vs. 7.3±0.9; p < 0.01). We next tested the ability of host aly/aly and aly/+ APCs to stimulate donor Balb/c T cells in vitro. No differences in proliferation, IFN γ production or CTL generation were detected, thus showing that the allo-stimulatory capacity of host APCs was not different between groups. In order to ascertain what extra-lymphoid host tissues might serve as initial sites for allo-antigen exposure, we examined donor T cell expansion (CD3+), activation (CD69+) and proliferation (CFSE) in the bone marrow compartment 3 days after BMT. We found that in each case, LN/PP/SP −/ − allo-BMT recipients had significantly higher numbers / divisions compared to allo, littermate, (LN/PP/SP +/+) controls. Collectively, these data challenge the paradigm that secondary lymphoid tissues are required for GVHD induction, and suggest that the bone marrow may represent an alternative site for allo-antigen recognition and donor T cell activation.

Targeting NF-Kβ for the Prevention of Graft Versus Host Disease.

NF-κB is a transcription factor that controls expression of a number of genes that are important for mediating immune and inflammatory responses, such as IL-1 and TNF-α , which are both overproduced during graft versus host disease (GVHD). To determine whether the NF-κB signaling pathway plays a role in the pathophysiology of GVHD, we examined the affect of bortezomib (PS-341, Velcade®), a proteasome inhibitor whose mechansism of action includes but is not limited to inhibition of NF-κB, and PS-1145, a IKB kinase (IKK) inhibitor that selectively inhibits NF-κB, on the induction of GVHD. Lethally irradiated C57BL/6 (H-2b) mice that were transplanted with MHC-incompatible B10.BR (H-2k) bone marrow (BM) and spleen cells and treated with a single dose of bortezomib (1 mg/kg) on the day of BMT had significantly prolonged survival compared to untreated GVHD controls (50% versus 0% survival at 60 days). In contrast, extended or delayed administration of this agent on a twice weekly schedule that recapitulated current clinical practice guidelines resulted in a significant increase in mortality as all animals died within ten days of transplantation. Histological analysis of bortezomib-treated mice demonstrated that mortality was attributable to severe pathological damage in the colon, indicating that there was a narrow therapeutic window within which bortezomib could be administered. Toxicity was GVHD-dependent as syngeneic marrow transplant recipients treated with a similar schedule for four weeks (8 doses) all survived for 60 days and had no evidence of pathological damage when compared to vehicle-treated controls. Given the narrow therapeutic window observed with bortezomib, we examined whether more selective inhibition of NF-κB with PS-1145 could prevent GVHD without inducing toxicity and early mortality. In vitro studies demonstrated that PS-1145 induced apoptosis in alloactivated T cells, similar to bortezomib, although a higher concentration was required for equivalent results (8 μM versus 0.32 μM). To achieve a serum concentration of PS-1145 necessary for the induction of T cell apoptosis in vivo, PS-1145 was administered at a dose of 50 mg/kg on days 0–2 post transplantation to recipients of MHC-incompatible marrow grafts. Treatment with PS-1145 resulted in similar GVHD protection when compared to animals administered a single dose of bortezomib. Prevention of GVHD by bortezomib and PS-1145 was associated with equivalent reductions of serum cytokine levels of IL-2, IL-10 and TNF-α when compared to GVHD controls. To determine whether the selective inhibition of NF-κB with PS-1145 caused less toxicity in BMT recipients, mice were administered this agent for 10 consecutive days beginning on the day of transplant. In contrast to results observed with bortezomib, PS-1145-treated mice had no early mortality and had superior survival when compared to GVHD controls (100% versus 0% survival at 60 days). GVHD protection in these mice was associated with significantly reduced expansion of alloreactive donor T cells in the spleen without compromise of donor engraftment. These results confirm a critical role for NF-κB in the pathophysiology of GVHD and indicate that selective inhibition of NF-κB may have a superior therapeutic index and constitute a viable approach to reduce GVHD.

Cerebral endothelial expression of adhesion molecules in mice with chronic graft-versus-host disease.

Graft-versus-host disease (GvHD) is a major complication after allogeneic bone marrow transplantation (BMT). The theory that the central nervous system (in addition to the peripheral nervous system) participates in GvHD has been supported by detection of cerebral lymphomononuclear infiltrates in experimental GvHD and the observation of cerebral angiitis-like disease in patients with chronic GvHD. In a murine BMT model, we investigated the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on cerebral endothelium after allogeneic and syngeneic transplantation. Through the use of ICAM-1-knockout mice, the effect of ICAM-1 deficiency on cellular infiltration was evaluated. As an indicator of enhanced apoptotic cell death, we examined the cerebral expression of Fas antigen (Fas), the occurrence of the poly-ADP ribose polymerase p85 fragment, and the distribution of TUNEL positive-stained cells. In close correlation with earlier findings of cerebral infiltration in the same animals, we found cerebral endothelial upregulation of ICAM-1 and especially of VCAM-1 in allogeneic recipients compared with syngeneic animals without GvHD and unmanipulated controls. In ICAM-1-knockout mice, leukocytic infiltration did not differ from that in wild-type animals. Neither cerebral histopathologic changes nor an apoptotic effect of cellular infiltrates on brain parenchyma could be detected. In this model of experimental GvHD, VCAM-1 may play a critical role in leukocyte recruitment into the central nervous system of animals with chronic GvHD.

Selective elimination of alloreactive donor T cells attenuates graft-versus-host disease and enhances T-cell reconstitution

Impaired T-cell immune reconstitution is a major complication after allogeneic bone marrow transplantation (BMT) and is particularly exacerbated in the setting of graft-versus-host disease (GVHD). Conventional approaches to reduce GVHD, such as T-cell depletion or pharmacologic immunosuppression, typically fail to enhance T-cell immunity and often further exacerbate this problem. An alternative strategy to mitigate GVHD severity is the selective elimination of graft-versus-host-reactive donor T cells by using an incorporated thymidine kinase suicide gene. This approach has been shown to effectively reduce GVHD, although the effect of this strategy on T-cell reconstitution is unresolved. We addressed this question in a murine BMT model (C57BL/6 [H-2 b] → AKR/J [H-2 k]) in which donor and recipient differ at major and minor histocompatibility antigens. Lethally irradiated AKR recipients transplanted with T cell-depleted bone marrow plus thymidine kinase-positive T cells followed by post-BMT ganciclovir (GCV) administration had more prompt and complete normalization of the T-cell repertoire than phosphate-buffered saline-treated GVHD control animals. By 60 days after transplantation, mice administered GCV had T-cell repertoires that were virtually indistinguishable from those of mice that underwent transplantation with T cell-depleted bone marrow alone (no GVHD controls) when assayed by T-cell receptor (TCR) spectratyping. In contrast, phosphate-buffered saline-treated animals had persistent skewing in most Vβ families. T cells obtained from GCV-treated mice also had significantly higher in vitro proliferative responses after posttransplantation inoculation with ovalbumin than GVHD animals, indicating that CD4 + T-cell responses against a nominal antigen were better preserved in these chimeras. Finally, GCV-treated mice had augmented immune reconstitution in response to exogenous interleukin-7 administration, as evidenced by increased overall spleen cellularity and absolute numbers of T and B cells. This was in contrast to GVHD control animals, which had a blunted response to interleukin-7 administration. These data indicate that GVHD severity can be significantly reduced by selective elimination of alloreactive donor T cells without compromise of T-cell immunity. Moreover, in light of previous studies demonstrating that this strategy can reduce GVHD without loss of alloengraftment and antileukemia reactivity, further examination of this approach in humans seems warranted.

Interleukin-12 Inhibits Graft-Versus-Host Disease Through an Fas-Mediated Mechanism Associated With Alterations in Donor T-Cell Activation and Expansion

AbstractWe have recently made the paradoxical observation that a single injection of recombinant murine interleukin-12 (IL-12) on the day of bone marrow transplantation (BMT) inhibits graft-versus-host disease (GVHD) in lethally irradiated mice receiving fully major histocompatability complex (MHC)-mismatched bone marrow and spleen cells. We have now examined the mechanism of this effect of IL-12 on acute GVHD. By day 4 post-BMT, IL-12–treated mice showed marked reductions in splenic donor CD4+and CD8+T cells compared with GVHD controls. Expression of the early activation markers IL-2R alpha chain (CD25) and CD69 on splenic donor CD4+cells was considerably higher at early time points (36 and 72 hours post-BMT) in IL-12–treated mice compared with GVHD controls. However, the later, GVHD-associated increase in CD25 and very late antigen-4 (VLA-4) expression on donor T cells was greatly depressed in IL-12–protected mice compared with GVHD controls. The marked GVHD-associated expansion of host-reactive T helper cells by day 4 was also completely inhibited in the IL-12–treated group. Expression of Fas was increased on donor CD4 cells of IL-12–treated mice compared with those of controls on days 3 through 7 post-BMT. Furthermore, the ability of IL-12 to protect against GVHD was at least partially dependent on the ability of donor cells to express functional Fas molecules. We conclude that IL-12 treatment at the time of BMT markedly perturbs the activation of alloreactive donor CD4+T cells that play a critical role in the pathogenesis of acute GVHD. We hypothesize that these perturbations culminate in Fas-dependent apoptosis of donor T cells, thus impeding their expansion and their GVHD-promoting activity.

Interleukin-12 Inhibits Graft-Versus-Host Disease Through an Fas-Mediated Mechanism Associated With Alterations in Donor T-Cell Activation and Expansion

We have recently made the paradoxical observation that a single injection of recombinant murine interleukin-12 (IL-12) on the day of bone marrow transplantation (BMT) inhibits graft-versus-host disease (GVHD) in lethally irradiated mice receiving fully major histocompatability complex (MHC)-mismatched bone marrow and spleen cells. We have now examined the mechanism of this effect of IL-12 on acute GVHD. By day 4 post-BMT, IL-12–treated mice showed marked reductions in splenic donor CD4+ and CD8+ T cells compared with GVHD controls. Expression of the early activation markers IL-2R alpha chain (CD25) and CD69 on splenic donor CD4+ cells was considerably higher at early time points (36 and 72 hours post-BMT) in IL-12–treated mice compared with GVHD controls. However, the later, GVHD-associated increase in CD25 and very late antigen-4 (VLA-4) expression on donor T cells was greatly depressed in IL-12–protected mice compared with GVHD controls. The marked GVHD-associated expansion of host-reactive T helper cells by day 4 was also completely inhibited in the IL-12–treated group. Expression of Fas was increased on donor CD4 cells of IL-12–treated mice compared with those of controls on days 3 through 7 post-BMT. Furthermore, the ability of IL-12 to protect against GVHD was at least partially dependent on the ability of donor cells to express functional Fas molecules. We conclude that IL-12 treatment at the time of BMT markedly perturbs the activation of alloreactive donor CD4+ T cells that play a critical role in the pathogenesis of acute GVHD. We hypothesize that these perturbations culminate in Fas-dependent apoptosis of donor T cells, thus impeding their expansion and their GVHD-promoting activity.

Interleukin-12 Inhibits Murine Graft-Versus-Host Disease

Interleukin-12 (IL-12) is a potent immunostimulatory cytokine and an inducer of type-1 T-helper cell activity and of cytotoxic T lymphocyte and natural killer cell function. We report here the paradoxical observation that a single injection of 4,900 IU of recombinant murine IL-12 inhibits acute graft-versus-host disease (GVHD) in a fully major histocompatibility complex (MHC) plus multiple minor antigen-mismatched bone marrow transplantation (BMT) model (A/J-->B10). The protective effect was enhanced by administration of T-cell-depleted host-type BM cells, and complete donor-type lymphohematopoietic reconstitution was observed in most animals. Treatment with a protective course of IL-12 led to increased serum interferon-gamma (IFN-gamma) levels as compared with those for GVHD controls at early time points, when IFN-gamma was produced predominantly by host-type natural killer cells, but led to almost complete inhibition of the later GVHD-associated increase in serum IFN-gamma levels, when IFN-gamma is produced predominantly by CD4+ T cells. Furthermore, IL-12 treatment was associated with marked alterations in the kinetics of donor T-cell expansion. Reductions in donor CD4+ and CD8+ T cells were observed in the spleen on day 4 post-BMT, but a marked increase in donor CD8+ cells was observed on day 7. Unlike broadly immunosuppressive methods for inhibiting GVHD, which are associated with loss of antileukemic effects, IL-12 has the potential to mediate antileukemic effects of its own; therefore, the GVHD-inhibitory effects of IL-12 described here suggest a potential application for this cytokine in clinical BMT.

Inhibition of graft-versus-host disease by interleukin-2 treatment is associated with altered cytokine production by expanded graft-versus-host-reactive CD4+ helper cells.

In a fully MHC plus multiple minor antigen-mismatched murine bone marrow transplantation (BMT) model, we have demonstrated that a short course of high dose IL-2, begun on the day of BMT, protects against graft-versus-host disease (GVHD). This inhibitory effect is directed against donor CD4+ cells. To determine whether the mechanism of IL-2-induced GVHD protection involves clonal deletion or anergy of host-reactive donor T helper cells (Th), we performed limiting dilution analyses to measure the frequency of activated Th that reacted to donor, host, and third-party antigens in GVHD control and IL-2-protected mice. Marked and specific expansion of host-reactive Th was observed to a similar extent in GVHD control and IL-2-protected mice by day 5 after BMT, and the number of these cells in the spleen increased by several orders of magnitude between days 3 and 5 after BMT, which suggests that recirculation from other tissues occurred in this period. A high proportion (approximately 80%) of donor T cells expressed CD25 in both GVHD control and IL-2-protected mice on day 4 after BMT, which suggests a high level of bystander T cell activation. Since marked quantitative differences in the GVH response were not observed between GVHD control and IL-2-protected mice, we assessed both groups for qualitative differences in the Th response. Spleen cells isolated in the first 8 days after BMT were cultured with host-type, donor-type, or third-party stimulators or without stimulators, and cytokines were measured in supernatants harvested at 24 hr. GVHD was associated with marked increases in supernatant IFN-gamma levels from day 3 to day 6 after BMT, and with increases in IL-2 levels compared with naive A/J controls or syngeneic BMT controls stimulated with host antigens. Production of these cytokines was specifically induced by host-type antigens. Supernatants from spleens of IL-2-treated mice showed delayed kinetics of IFN-gamma production, and tended to contain higher levels of IL-4 in response to host antigen compared with GVHD controls on days 2 and 4 after BMT. Both IL-4 and IFN-gamma were produced almost exclusively by CD4+ cells in spleens of GVHD control and IL-2-protected mice on day 4. However, no consistent difference was observed between the groups in supernatant IL-2 or IL-10 levels, ruling out a simple Th1 to Th2 switch.(ABSTRACT TRUNCATED AT 400 WORDS)

Amelioration of acute GVHD disease and reestablishment of tolerance by short term treatment with anti TCR antibody

We investigated whether tolerance can be reestablished in mice with graft-versus-host disease (GVHD) by using a short-term, T-celldepleting treatment with an anti-TCR-αβ mAb. GVHD was induced in 950-rad—irradiated AKR mice (H-2k, Mls-1a) by injecting 5 × 106 T celldepleted bone marrow cells together with either 107 or 2 × 106 lymph node (LN) cells from BALB/c mice (H-2d, and Mls-1b). AKR mice that received 107 LN cells exhibited a severe form of acute GVHD, in which all mice died by day 60. In this severe form of GVHD, treatment with anti-TCR-αβ-mAb completely ameliorated the induction of GVHD when initiated on day 0 (a total of 800 μg/mouse administered on days 0, 5, and 10). When the same protocol was begun on day 10, it had no therapeutic effect. However, this delayed treatment with anti-TCR-αβ mAb was very effective in reversing a less severe form of GVHD that was induced by the injection of 2 × 105 donor LN cells. Recipient mice given prophylactic antiTCR-αβ treatment achieved host-specific tolerance in association with clonal deletion of host Mls-1a-reactive Vβ+T cells. In contrast, spleen cells from recipient mice that recovered from the mild form of GVHD as a result of the delayed anti-TCR-αβ treatment contained a considerable proportion of Vβ6+T cells, despite the healthy appearance of these mice. A MLR assay showed that the spleen cells from these mice responded well to Mls-1a Ag but not to H-2k Ag, in contrast with the apparent responses of spleen cells from untreated GVHD controls to both Ags. In addition, cells from the anti-TCR-αβ treated mice exhibited a specific reduction in cytotoxicity against AKR blasts. Collectively, these data indicated that a short-term treatment of mice having GVHD with an anti-TCR-αβ mAb, starting even after disease onset, can re-establish host-specific tolerance, at least to the host-histocompatibility Ag. (Copyright 1994. The Journal of Immunology.)