Signs Of Graft-versus-host Disease Research Articles

Mild cold stress resulting from standard housing conditions for laboratory mice masks the severity of graft vs. host disease in mouse models of bone marrow transplantation (TRAN3P.887)

Abstract Clinical bone marrow transplants (BMT) yield lethal graft vs. host disease (GVHD) after transplant of un-manipulated bone marrow (BM). This differs from murine GVHD models that typically require addition of spleen or lymph node derived T cells. Recent work has shown that mice housed at mandated sub-thermoneutral ambient temperatures (22°C) are cold stressed, resulting in heat generation activated by the sympathetic nervous system. Since cold stressed mice exhibit weakened immune responses, we hypothesize that baseline GVHD is reduced compared to that seen in mice at thermoneutral temperatures (30°C). We compared GVHD in mice receiving MHC-mismatched un-manipulated BM while housed at 22 vs. 30°C at which cold stress is alleviated. In this BMT model, mice housed at 22°C show no signs of GVHD, however, we found weight loss and lethal GVHD in mice housed at 30°C. The same trend was observed with MHC-matched allogeneic BMT. As housing at 22°C activates the sympathetic nervous system, we blocked its activation by administering propranolol, a commonly prescribed β-blocker. Following BMT, mice at 22°C treated with β-blocker displayed lethal GVHD similar to untreated mice at 30°C. This suggests that T cell responses are dampened by cold stress through a β-adrenergic pathway which is circumvented by thermoneutral housing. This study provides insight into translational discrepancies of clinical and murine BMT and demonstrates lethal GVHD in a mouse model generated by un-manipulated BM.

Alpha/Beta T-Cell Depleted Grafts as an Immunological Booster to Treat Graft Failure after Hematopoietic Stem Cell Transplantation with HLA-Matched Related and Unrelated Donors

Allogeneic hematopoietic stem cell transplantation is associated with several complications and risk factors, for example, graft versus host disease (GVHD), viral infections, relapse, and graft rejection. While high levels of CD3+ cells in grafts can contribute to GVHD, they also promote the graft versus leukemia (GVL) effect. Infusions of extra lymphocytes from the original stem cell donor can be used as a treatment after transplantation for relapse or poor immune reconstitution but also they increase the risk for GVHD. In peripheral blood, 95% of T-cells express the αβ T-cell receptor and the remaining T-cells express the γδ T-cell receptor. As αβ T-cells are the primary mediators of GVHD, depleting them from the graft should reduce this risk. In this pilot study, five patients transplanted with HLA-matched related and unrelated donors were treated with αβ T-cell depleted stem cell boosts. The majority of γδ T-cells in the grafts expressed Vδ2 and/or Vγ9. Most patients receiving αβ-depleted stem cell boosts increased their levels of white blood cells, platelets, and/or granulocytes 30 days after infusion. No signs of GVHD or other side effects were detected. A larger pool of patients with longer follow-up time is needed to confirm the data in this study.

Recruitment Of T Cells and Macrophages To The Eyes In Recipients Of Allogeneic Hematopoietic Stem Cell Transplants Correlate With Inflammatory Cytokine Presence In Ocular Gvhd

Allogeneic hematopoietic stem cell transplantation (HSCT) has become the standard of care for the treatment of several life-threatening hematologic malignancies as well as certain immunodeficiency disorders. Unfortunately, as the survival rate of patients with these diseases is improved, the quality of life is negatively impacted by the development of Graft vs. Host Disease (GVHD). GVHD is a complex, multi-organ disorder arising from an immunological attack by donor allo-reactive T cells that results in damage to vital organs including the liver, skin, hematopoietic compartment and the ocular surface of the eye. Ocular GVHD occurs in >60% of these patients and is characterized by dry eye, conjunctiva damage, punctate keratopathy, corneal ulceration and perforation. Despite the high frequency of eye involvement in patients undergoing GVHD, little is known regarding the underlying immune mechanisms responsible for ocular GVHD, limiting the ophthalmic care of these patients to palliative therapies and global anti-inflammatory drugs. In this study, we examined the ocular and immunological changes occurring in recipients of MHC-matched, minor antigen mis-matched donor HSCT. C3H.SW (H-2b, Ly9.1+) mice transplanted with EGFP+ B6 (H-2b, Ly9.1-) T cell depleted bone marrow cells (TCD-BM) supplemented with T cells: a) underwent weight loss and began exhibiting clinical signs of GVHD ∼3wks post-HSCT, b) contained damaged thymuses, c) expressed an inverted CD4/CD8 ratio in the peripheral lymphoid compartments, d) contained activated effector cells and e) low CD19 levels. Importantly, these mice also developed ocular surface disease evidenced by progression of ocular surface damage characterized by increased corneal fluorescein staining and ulceration by week 6 (Figure). Furthermore, histological analyses demonstrated that only mice that developed systemic GVHD exhibited corneal thickening and epithelial irregularity. Ocular pathology was also associated with conjunctiva involvement indicated by significant goblet cell destruction as well as dense inflammatory cell infiltrates identified by intra-vital fluorescent microscopy (Figure). IHC and flow analyses demonstrated donor EGFP+ Ly9.1- CD4+ and CD8+ T cells. Notably, significant levels of Ly9.1- EGFP-CD11b+ macrophages had also infiltrated the ocular surface. In contrast to the systemic CD8>CD4 GVHD associated phenotype, the T cell infiltrate in the ocular compartment was reversed; i.e. CD8<CD4. We detected IFNγ and TNFα mRNA from corneal tissue, which is consistent with Th1 effector allo-reactive cells and M1 inflammatory macrophages involvement in ocular GVHD. In total, the present findings have identified alterations and pathology in the eye and adnexa reflective of ocular GVHD and unequivocally demonstrate the presence of donor T cells in the ocular surface. We hypothesize that T cell–macrophage interactions underlie the pathology detected in this pre-clinical model and studies are underway to develop local therapeutic modalities targeting these infiltrative populations. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Prophylactic G-CSF mobilized donor lymphocytes infusion after non-myeloablative stem cell transplantation prevents relapse in patients with high-risk leukemia

To evaluate the anti-leukemia effects of prophylactic G-CSF mobilized donor lymphocytes infusion (pG-DLI) and its relationship with the incidence of graft-versus-host disease (GVHD) in high-risk leukemia patients with non-myeloablative stem cell transplantation (NST). 12 patients with high-risk leukemia were analyzed, including Ph⁺ acute lymphocytic leukemia (n=1), acute leukemia (AL) with persistent non-complete remission (n=2), acute myeloid leukemia (AML) with central nervous system (CNS) relapse (n=3), hybrid AL (n=1), secondary AML evolving from myelodysplastic syndrome (MDS/AML) (n=2), chronic myeloid leukemia in accelerated phase (CML-AP) (n=1), CML in blastic phase (CML-BP) (n=2). All patients received non-myeloablative conditioning and pG-DLIs were administered 30-40 days post transplantation if no signs of GVHD were present. The percentage of donor cell chimera was analyzed by short tandem repeat-polymerase chain reaction (STR-PCR) just before and after pG-DLI. The incidence of leukemia relapse and GVHD were observed. 12 high-risk leukemia patients with a median age of 38 (range: 29-52) years received G-DLI at a median interval of 35 (32-40) days. The median numbers of infused mononuclear cells (MNCs), CD34⁺, and CD3⁺ cells/kg recipient body weight was 2.3×10⁸/kg, 1.7×10⁶/kg, and 0.6×10⁸/kg, respectively. 10 of 12 patients had full donor chimera before pG-DLIs and conversion from mixed to full donor chimera occurred in the other 2 patients shortly after pG-DLI. Grade II acute GVHD (aGVHD) was observed in only 2 patients and chronic GVHD (cGVHD) developed in 6 patients, including involvement of skin (n=3), skin and intestine (n=2), liver (n=1). 1 patient died of cGVHD. With a median follow-up of 40 (24-64) months, 7 patients are alive in remission, with 3-year actuarial overall survival (OS) and disease-free survival (DFS) rates of the same 58.3%. Our findings indicate that pG-DLI after NST does not increase the risk of aGVHD, but could enhance the capacity graft-vs-leukemia and prevent relapse in high-risk leukemia patients.

Alterations in Chemokine Receptor CCR5 Expression on Blood Dendritic Cells Correlate With Acute Graft-Versus-Host Disease

Dendritic cells (DC) are important in the development of acute graft-versus-host disease (GVHD) after allogeneic hemopoietic cell transplantation (alloHCT). The trafficking of immature DC from blood to GVHD target organs is likely to be regulated by chemokine receptors. We performed flow cytometry to document the expression of chemokine receptors on circulating DC and correlated the findings after alloHCT with occurrence of acute GVHD. In normal individuals, plasmacytoid DC (pDC) expressed high levels of CCR5, whereas the major CD16 myeloid DC subpopulation lacked CCR5. However, its expression on CD16 cells was induced by culture in allogeneic mixed lymphocyte reaction supernatant, an effect largely mediated by interferon-γ. CCR5 was expressed on a significant proportion of CD16 DC in 42 alloHCT patients, whereas it was down-regulated on pDC. The maximum percentage of CCR5CD16 DC, at any time after transplantation, correlated with acute GVHD, whereas the minimum CCR5 on pDC showed a similar correlation. Before developing signs of GVHD, the maximum percentage CCR5CD16 DC was higher in patients with GVHD grades II to IV than in GVHD grades 0 and I, whereas the minimum percentage CCR5 on pDC was lower in GVHD grades II to IV than in GVHD grades 0 and I. CCR5 levels more than 20.5% on CD16 myeloid DC and less than 22.6% on CD123 pDC correlated with subsequent GVHD grades II to IV with high sensitivities and specificities. These observations may reflect DC activation and altered homing during the alloimmune response and could allow early diagnosis and therapeutic intervention before the clinical diagnosis of GVHD.

CD30-Targeted Therapy with Brentuximab Vedotin and DLI in a Patient with T-Cell Posttransplantation Lymphoma

CD30-Targeted Therapy with Brentuximab Vedotin and DLI in a Patient with T-Cell Posttransplantation Lymphoma

Infusion of clinical‐grade enriched regulatory T cells delays experimental xenogeneic graft‐versus‐host disease

We investigated the ability of clinical-grade enriched human regulatory T cells (Treg) to attenuate experimental xenogeneic graft-versus-host disease (GVHD) induced by peripheral blood mononuclear cells (PBMNCs; autologous to Treg) infusion in NSG mice, as well as verified their inability to induce xenogeneic GVHD when infused alone. Human Treg were isolated from peripheral blood apheresis products with a cell separation system (CliniMACS, Miltenyi Biotec GmbH) using a two-step procedure (simultaneous CD8 and CD19 depletion followed by CD25-positive selection) in six independent experiments with six different healthy volunteer donors. Sublethally (2.5 Gy) irradiated NSG mice were given 2 × 10(6) cytapheresis (PBMNC) product cells intravenously (IV) without (PBMNC group) or with 1 × 10(6) Treg (PBMNC + Treg group), while other NSG mice received 2 × 10(6) enriched Treg alone (also in IV; Treg group). The first five procedures were successful at obtaining a relatively pure Treg population (defined as >50%), while the sixth procedure, due to a technical problem, was not (Treg purity, 42%). Treg cotransfusion significantly delayed death from xenogeneic GVHD in the first five experiments, (p < 0.0001) but not in the sixth experiment. Importantly, none of the mice given enriched Treg alone (Treg group) experienced clinical signs of GVHD, while, interestingly, the CD4+ cells found in these mice 26 days after transplantation were mainly conventional T cells (median CD25+FoxP3+ cells among human CD4+ total cells were only 2.1, 3.1, and 12.2% in spleen, marrow, and blood, respectively). Infusion of clinical-grade enriched Treg delayed the occurrence of xenogeneic GVHD without inducing toxicity in this murine model.

Inhibition of Jak1/Jak2 Is More Effective Than Inhibition of Jak3 in Protecting Mice From Acute Graft-Versus-Host Disease (aGvHD) by Significantly Decreasing Alloreactive CD4+ T-Cells.

AbstractAbstract 2997▪▪This icon denotes a clinically relevant abstract Background:Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment for patients with hematological malignancies. However, its success is limited by a life-threatening complication: the graft-versus-host disease (GvHD). Although numerous studies have described immunosuppression protocols to mitigate acute GVHD (aGvHD), novel approaches are needed. Chemokines are well known inducers of leukocyte trafficking and activation. Stimulation of the chemokine receptor signaling pathway leads to initiation of the Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway that contributes to the pathogenesis of GvHD. The key role of JAK signaling in normal and abnormal lymphocyte development and function, along with the cytotoxic effects of its inhibitor INCB18424 (Ruxolitinib) on leukemia cells, prompted us to hypothesize that this selective JAK1 and 2 inhibitor could be useful as anti-GvHD agent while maintaining antitumor activity. Since CP-690550, a more selective JAK3 inhibitor, was recently shown to protect against GvHD in mouse models, we also tested whether blocking the JAK1/JAK2 pathway could be more effective in preventing GvHD. Methods:To assess the therapeutic effect of pharmacologic modulation of JAK1 and 2 on GvHD, a major histocompatibility complex (MHC) mismatched HSCT mouse model was used. Recipient BALB/c mice were lethally irradiated and treated either with spleen and bone marrow (BM) cells from C57BL/6 (B6) donors (GvHD cohort, n=8), or with spleen and BM cells from B6 donors along with INCB18424 90mg/kg/day at days -1 to 13 (INCB18424 cohort, n=10) or with CP-690550 15mg/kg/day (CP-690550 cohort, n=8) at days -1 to 13. Syngeneic transplants (B6-B6, n=6) and BALB/c recipients treated with B6 BM cells only (control cohort, n=8) were also included as controls. Mice were characterized for GvHD by monitoring overall survival and weight loss. Recipient mice were sacrificed and tissues harvested on day 14 and 30 post transplant and GvHD confirmed by histology. Results:All mice in the GvHD cohort had clinical evidence of GvHD (weight loss, generalized erythema of the skin and poor fur quality) by day 14. The INCB18424 treated mice showed markedly reduced weight loss along the time of observation when compared to the GvHD cohort. Animals in the CP-690550 cohort tended to gain weight during the time of treatment (day-1 to 13), but thereafter they exhibited reduced body weight similar to that observed in the GvHD cohort. The histological examination of the stomach, liver, skin and intestine obtained at day 14 revealed no sign of GvHD in the control group as well as in the INCB18424 group. On the other hand, mild to moderate signs of GvHD were present in the tissues of CP-690550 treated mice and extensive inflammation and disruption of the normal architecture of the tissues was observed in the GvHD group. To determine whether INCB18424 treatment affected alloreactive CD4+ T cells, total spleen T cells were harvested at day 14 from the GvHD cohort and from recipients either of INCB18424 or CP-690550. Total spleen T cells were co-cultured with BM derived BALB/c (recipient-derived) or C57BL/6 (donor-derived) dendritic cells (DCs). After 24h, T cells alloreactivity was determined by IFN-γ production assessed by intracellular staining. As expected, T cells from GvHD mice showed significantly higher alloreactivity against BALB/c DCs compared to the reactivity observed against syngeneic B6 DCs (5.24% and 0.84% respectively, p<0.05). The alloreactivity observed when T cells from INCB18424 treated mice were stimulated with allogeneic BALB/c DCs was significantly lower than that in the GvHD group (0.64% and 5.24% respectively, p<0.05) and eas also lower than that of the CP-690550 group (1.43%). STAT phosphorylation analysis demonstrated that INCB18424 treatment was effective in vitro. Conclusions:The inhibition of Jak/STAT signaling using the sensitive and specific inhibitor of Jak1/Jak2, INCB18424, conferred effective protection from aGvHD in a HSCT mouse model. INCB18424 treatment was more effective than the targeting of JAK3 with CP-690550. In fact, CP-690550 administered during GvHD induction was not completely sufficient to restore the normal weight and to prevent the histological appearance of GvHD whereas INCB18424 was. INCB18424 protected mice against acute GvHD by significantly decreasing alloreactive CD4 T cells. Disclosures:No relevant conflicts of interest to declare.

A Preclinical Canine Model of in Utero Hematopoietic Cell Transplantation–Induced Graft Vs. Host Disease.

AbstractAbstract 3002 Introduction:In utero hematopoietic cell transplantation (IUHCT) offers the potential to achieve allo-engraftment and associated donor-specific tolerance (DST) without toxic conditioning. To evaluate IUHCT in a clinically relevant large animal model, we have developed the haploidentical canine model of IUHCT. Recent optimization of the model using a CD3 depleted graft has achieved consistent and stable levels of mixed hematopoietic chimerism (average > 10%) without graft versus host disease (GVHD). However, since GVHD represents the greatest theoretical risk of clinical IUHCT, it is important to identify the phenotype and requirements for IUHCT-induced GVHD in the canine model prior to clinical application. In the current study, we attempted to establish a threshold dose of CD3+ cells required for IUHCT-induced GVHD in the haploidentical canine model by increasing the absolute percentage of CD3+ cells in the donor inoculum and we characterized the GVHD phenotype observed. Methods:43 fetuses of 9 pregnant bitches underwent ultrasound-guided intracardiac injection of maternal BM cells at 37.5–43 days gestational age (GA). In our large animal model, bone marrow yields, cellularity and litter size differ between dams. Due to these factors and because the targeted gestational age range coincides with an exponential growth phase for the dog fetus, standardization of cell dose per weight was not possible. Following MACS CD3 depletion, graft composition was standardized by adding back the CD3+ fraction to achieve donor CD3 content of 1%, 3%, 7.5%, and 16% (whole bone marrow [WBM] positive control) (Table 1). Live born puppies were monitored daily for signs of GVHD, including skin lesions, diarrhea, hematochezia, jaundice, weight loss and lethargy. Chimerism was measured by a PCR-based VNTR assay using commercially available primers. Results:Though prenatal survival of 75% was not significantly different than baseline (76%) and pups were not runted at birth, acute GVHD was observed postnatally in all ten live born recipients of either 7.5% CD3 cells or WBM, with onset ranging from 1–14 days of age. Recipients of grafts containing 1% and 3% CD3 cells showed no clinical evidence of GVHD.Weight loss was the earliest and most consistent clinical feature of IUHCT-induced GVHD in our model. Various affected animals manifested additional symptoms in all GVHD target organs, including increased respiratory rate/effort, hepatic dysfunction, bloody diarrhea and skin lesions. The phenotype was severe and all ten ultimately required euthanasia between 2 and 21 days of age.Necropsies performed during acute GVHD revealed significant erythematous and/or ulcerated skin change, hepatosplenomegaly, and punctate hemorrhage throughout the gastrointestinal tract and lung. Histology (H&E) demonstrated lymphocytic infiltrates and focal necrosis in target tissues as well as effacement of the dermal-epidermal junction and villous surfaces. Canalicular bile stasis was the most uniform and characteristic microscopic finding irrespective of hepatic functional status.Chimerism levels in pups who survived to birth were compared based upon CD3 content of the donor graft (Table 1). Increasing CD3 percentage trended toward an increase in chimerism, though only the WBM group reached statistical significance (p=.043). Conclusions:In the preclinical haploidentical canine model of IUHCT the threshold for induction of GVHD appears to be between 3% and 7.5% graft CD3+cell content. Interestingly, the absolute dose of CD3+ cells/kg fetal weight does not entirely correlate with the occurrence of GVHD, suggesting that the composition of the graft rather than the absolute T-cell content may determine the likelihood of GVHD. Given the complete absence of GVHD at a CD3 dose of 1% and the excellent engraftment achieved, it appears that there is a safe therapeutic index for achievement of chimerism without GVHD. When IUHCT-induced GVHD occurs, it is severe and clinically similar to postnatal GVHD. Notably, clinical GVHD only occurred after birth, suggesting suppressive mechanisms in utero. Additional studies are needed to further optimize the graft for support of engraftment without GVHD.Table 1DosenGA at IUHCTCD3 cells/kgCD34 cells/kgMean ChimerismGVHD1%2039–41.37–2.72 × 10 (9)5.8–58.2 × 10 (8)12.1%N3%4381.26 × 10 (9)9.8 × 10 (8)7.3%N7.5%341.52.26 × 10 (9)7.0 × 10 (8)17.7%Y16% (WBM)7432.0 × 10 (9)5.1 × 10 (8)20.3%Y Disclosures:No relevant conflicts of interest to declare.

G-CSF Stem Cell Mobilization Is Protective Against Acute Graft-Versus-Host Disease (aGvHD) by Increasing Myeloid-Derived Suppressor Cell (MDSC) Frequencies

AbstractAbstract 1898 Background:In mice, graft-versus-host disease (GvHD) can be abrogated by ex vivo expanded, bone marrow derived myeloid-derived suppressor cells (MDSCs) generated in the presence of GM-CSF, G-CSF and IL-13 (Highfill et al). Whether MDSCs play a role in human allogeneic hematopoietic stem cell transplantation (allo-HSCT) is still unclear. We hypothesized that G-CSF stem cell mobilization may be protective from acute GvHD (aGvHD) by increasing MDSC frequencies. Methods:G-CSF-mobilized peripheral blood (PB) samples were collected from 40 healthy unrelated donors (median age 34, range 20–43, male/female 31/9) who received G-CSF (Filgrastim) at 10 μg/kg/day for 5 days. Donor selection had been performed according to standard criteria, including molecular typing for HLA-A,-B,-C, DRB1, and DQB1. Donors were 10/10 HLA-matched (MUD) in 20 cases, 9/10 in 13 cases and 8/10 in the remaining 7 cases (MMUD). Patients (median age 46, range 18–67) received reduced intensity conditioning based or low-dose total body irradiation (TBI 2Gy) (8), Fludarabine-Rabbit Antithymocyte Globulin (ATG) (9) or Thiotepa-ATG (23). Diagnosis were lymphomas (29), myelomas (8), acute myeloid leukemia (3). GvHD prophylaxis was cyclosporine plus either methotrexate (36) or mycophenolate mofetil (4). As controls, PB samples were collected from 10 healthy adults. Informed consent was obtained from all subjects. Cells were characterized using flow cytometry with Abs against CD3;CD14;CD16;CD19;CD20;CD56;CD11b;HLADR;CD33. The frequencies of MDSCs and T regulatory cells (Tregs, CD4+CD25+CD127-FoxP3+) in the grafts were correlated with the clinical characteristics and outcome of the 40 patients. To verify that G-CSF treatment increases the number of MDSCs that are transferred with the graft and prevent GvHD, a major histocompatibility complex (MHC) mismatched HSCT mouse model was also used. After lethal irradiation, recipient BALB/c mice received spleen and bone marrow (BM) cells from C57BL/6 (B6) donors (GvHD cohort, n=5) or BM cells only (negative control, n=3). To generate MDSC enriched allografts, donor mice were treated with G-CSF (5 μg/d for 5 days) and thereafter BM cells and spleen cells were transferred in recipient mice (MDSC cohort, n=5). Results:Expansion of MDSCs (Lin-/LoHLADR−CD33+CD11b+) in the PB of G-CSF-treated unrelated donors was found with respect to steady state control individuals (p< 0.03, Mann Whitney-U). Acute GvHD occurred in 16 of 40 patients (40%). There was no significant correlation between the incidence of aGvHD and the degree of HLA incompatibility or the presence of donor-recipient sex mismatches. Neither the conditioning regimens nor the GvHD prophylaxis had effect on risk of aGvHD. There was no correlation between the number of Tregs infused and the occurrence of GvHD.Conversely, aGvHD patients received grafts containing significantly lower number of MDSCs when compared to non- aGvHD patients (p < 0.006, Mann Whitney-U). The ability of MDSC levels in the graft to predict the occurrence of aGvHD was determined by the receiver operating characteristic (ROC) curve: sensitivity was 100%, specificity 60%, AUC=0.768. The immunosuppressive activity of the MDSCs on activated T lymphocytes was confirmed in vitro. To confirm these results, we set up a MHC mismatched HSCT mouse model. G-CSF treatment induced a significant increase in MDSCs (up to four fold, p<0.05) whereas the frequencies of CD34+ and CD3+ cells were not affected. The MDSC-enriched BM cells transferred into lethally irradiated recipient mice together with spleen cells, resulted in a less severe aGvHD as confirmed by monitoring the clinical signs of GvHD (weight loss, generalized erythema of the skin, poor fur quality and overall survival) compared to the GvHD cohort not receiving G-CSF. Conclusions:G-CSF mobilization significantly increases circulating MDSC both in Matched Unrelated Donors and in mice. In humans, a significant correlation between the frequencies of MDSC present in the graft and the incidence of aGvHD was found. This result was further confirmed using a MHC mismatched HSCT mouse model. Mice treated with G-CSF received grafts containing higher numbers of MDSCs and developed less severe aGvHD than the untreated mice. Taken together, these findings strongly suggest that G-CSF stem cell mobilization is protective against aGvHD through MDSCs augmentation and might be relevant to modify GvHD prophylaxis. Disclosures:No relevant conflicts of interest to declare.

GSK3β Inhibition Promotes T Cell Reconstitution and Preserves Naive T Cell Phenotype in Bone Marrow Reconstituted Mice

AbstractAbstract 1890Stem cell transplantation has become a widely used procedure in the treatment of haematological and non-haematological clinical disorders. Unfortunately, cure is often hampered by relapse of the underlying disease, graft-versus-host disease (GVHD), or severe opportunistic infections. Slow T-cell reconstitution is regarded as primarily responsible for infections, GVHD, and relapse, therefore, enhancing immune reconstitution is important. Glycogen synthase kinase-3β (GSK3β) was recently identified as an important regulator of T cell function acting through the Wingless (Wnt) pathway. The effect of in vivo administration of GSK3β inhibitor 6-Bromoindirubin 3’-oxime (BIO) was examined in a humanised mouse model. Mice transplanted with highly purified cord blood CD34+ stem cells demonstrated efficient multilineage reconstitution including myeloid, B and T cell lymphoid compartments. The presence of human CD4 and CD8 single positive human T cells was abundant in peripheral blood (PB). De novo generated T cells exhibited low CD31 expression in the naïve CD4+ T cells suggesting prolonged post-thymic proliferative history of these cells. This is not completely surprising considering that graft recipient mice are characterized by impaired thymopoiesis following irradiation. Human T cells at various stages of differentiation including late effector T cells were recorded by detecting the expression of CD62L, CD45RA and CD45RO. Late memory T cell skewing was observed in PB and spleen of graft recipient mice. Activation of human T cells expressing CD25 was registered in the spleen, however, the recipients of the graft did not exhibit any signs of GVHD suggesting normal positive and negative selection occurring in the thymus during human T cell development in this mouse model. Human T cells isolated from the spleen of transplanted mice exhibited strong proliferative responses to mitogenic and allogeneic stimulation, however, they did not demonstrated any CTL activity tested following vaccination with human leukaemia U937 cells. In vivo administration of GSK3β inhibitor promoted T cell reconstitution in mice transplanted with human CD34+haematopoietic progenitor cells. Per cell output of T cells from CD34+ and CD34+CD38- primitive bone marrow (BM) progenitor cells was higher in BIO-treated mice while CD19+ B cell output was reduced suggesting T-cell developmental skewing in expense of B cell development. In vitro analysis of CD34+ progenitor cells co-cultured with bone marrow stroma MS5 cells has demonstrated inhibited B-cell development following BIO-treatment. CD31 expression in naïve CD4+ T cells was not up-regulated by BIO suggesting that GSK3β inhibition does not act to increase thymic output of T cells. GSK3β inhibition also increased naïve/memory T cell ratio in reconstituted mice. A similar effect was observed in mice transplanted with mature cord blood (CB)-derived T cells. delayed naive to memory T cell transition is likely related to decreased T cell activation and proliferation demonstrated ex vivo. BIO reduced IFNγ and TNFα production in human T cells. BIO increased naïve T cell production in mitogenically stimulated T cells and in mixed lymphocyte cultures. GSK3β inhibition preserved naïve T cell gene expression profile and suppressed the expression of genes activated during effector T cell differentiation. BIO actrivated β-catenin sigbnaling and up-regulated IL7Rα expression. IL7 signalling prevents activated T cell death following effector differentiation suggesting that the mechanism triggered by BIO may act through the inhibition of activated T cell death. In addition, BIO down-regulated negative regulator of IL7Rα SOCS1 as well as CTLA4 and PDCD1 both up-regulated during effector differentiation. Thus clinically GSK3β inhibition acting to prevent late memory T cell skewing and preserving a subset of naïve T cells may increase T cell diversity and improve T cell responses in the recipients of CB transplant particularly in adult patients with impaired thymic function. Disclosures:No relevant conflicts of interest to declare.

Induction of Graft-versus-host Disease and &lt;em&gt;In Vivo&lt;/em&gt; T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al. developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and &lt;em&gt;In Vivo&lt;/em&gt; T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Lenalidomide plus donor-lymphocytes infusion after allogeneic stem-cell transplantation with reduced-intensity conditioning in patients with high-risk multiple myeloma

Myeloma relapse is the main cause of death after allogeneic stem cell transplantation. The aim of our observational study was to evaluate the anti-myeloma effect of lenalidomide followed by donor-lymphocyte infusion (DLI) as post-transplantation adoptive immunotherapy. Twelve patients with refractory myeloma were analyzed. The median age at transplantation was 56 years (range, 46-64 years). All patients received reduced-intensity conditioning. Patients were included if progressive or residual disease was observed at day +100 and if no signs of graft-vs-host disease were evident. DLIs were administered after two cycles of lenalidomide. Median dose of lenalidomide was 15 mg (range, 10-25 mg). Patients received a median of six cycles (range, 1-10 cycles). Nine patients (60%) received an escalating dose of DLI. The 1 and 2-year probability of progression-free survival was 75% and 50%, and overall survival was 83% and 69%, respectively. Median overall survival was not reached and median progression-free survival was 23 months. Lenalidomide is well tolerated after allogeneic stem cell transplantation; the combination with DLI did not cause a higher risk of graft-vs-host disease; an immunological synergistic effect was probably present with this strategy. This combination should be evaluated further in a larger cohort of patients.

Long-Term Disease-Free Survival of Patients with AML Relapse After T-Cell-Depleted Allogeneic Stem Cell Transplantation by Re-Induction Therapy and Subsequent Interferon-Boosted Donor Lymphocyte Infusion

Abstract 3064Relapse of AML after allogeneic stem cell transplantation (alloSCT) has a very poor prognosis. Salvage re-induction chemotherapy leads to clinical remissions in a substantial number of patients, but these remissions tend to be of short duration. In contrast, donor lymphocyte infusions (DLI) have the potential to effect long-lasting remissions, but the interval of several weeks to months that is required to develop a DLI-induced anti-leukemia response may prevent efficient control of a highly proliferative leukemia. In addition, a high tumor burden may suppress the immune response. In contrast, the combination of efficient cytoreduction by chemotherapy with DLI administered in rapid succession under circumstances favoring the development of an early and profound immune response might have the potential to eradicate otherwise resistant leukemia cells. We therefore adopted an institutional therapeutic strategy for relapsed myeloid leukemia post-allogeneic SCT based on administration of DLI at the anticipated end of the neutropenic phase after salvage re-induction chemotherapy. At this time point, the high prevalence of a pro-inflammatory milieu should favor the induction of the immune response, and an expected state of lymphopenia should promote the expansion of infused T cells by homeostatic proliferation. If 3 weeks after DLI no graft versus host disease (GvHD) was observed, the potential anti-leukemia immune response was further amplified by treatment with interferon- α (IFN- α) until GvHD occurred.Between January 2000 and December 2009 44 patients with relapsed myeloid malignancy after alloSCT were treated at our hospital. Pre-transplant diagnoses were AML n=40, CMML n=1 and MDS n=3. Median time from SCT to relapse was 187 days. Median follow-up after relapse was 3.1 years. 5 patients had a smouldering relapse (<10% bone marrow blasts) and 39 patients had an overt relapse.Of 39 patients with overt relapse, 7 patients (18%) did not receive re-induction therapy due to poor performance status (n=5) or patient choice (n=2). 32 patients received remission-induction therapy consisting of gemtuzumab ozogamycin (n=9), cytosine arabinoside-containing chemotherapy (n=17), or both (n=6). Following this treatment, 7 of 32 patients had rapidly progressive disease during induction therapy (n=6) or died due to toxicity (n=1) and did not receive DLI.The remaining 25 patients received DLI at a dose of 5.0×10 ^6 CD3+ cells/kg for related and 2.5×10 ^6 CD3+ cells/kg for unrelated donors 3 weeks after the start of remission-induction therapy. In 16 of these patients DLI was boosted with IFN- α 3.0×10 ^6 IE once daily. This strategy resulted in acute GvHD in 17 of 25 patients (n=8 grade 1–2, n=9 grade 3–4). At 6 weeks after DLI, 16 patients had reached CR, 5 patients had failed to reach CR (2 with GvHD) and 4 suffered treatment-related mortality (3 with GvHD). Of the 16 patients in CR, 4 had no signs of GvHD and developed a second relapse during the follow-up period. Only 3 of 12 patients in CR with signs of acute GvHD at 6 weeks after DLI developed a second relapse. In total, 9 of 17 patients (53%) with acute GvHD after DLI had long term survival versus none without acute GvHD. During follow-up, 8 patients developed chronic GvHD (n=4 limited, n=4 extensive).Finally, 5 patients with an early detected smouldering relapse received DLI, which was boosted with IFN- α in 2 patients, without salvage re-induction therapy. All 5 patients developed GvHD (n=2 grade 1–2, n=3 grade 3–4) and 3 patients achieved a CR of whom 1 patient died from GvHD.Our results indicate that treatment of relapsed AML after alloSCT with salvage re-induction therapy followed by DLI at the end of the neutropenic phase during minimal residual disease, with additional boosting of the immune response with IFN- α, can result in long-term disease-free survival. Disclosures:Off Label Use: Interferon: DLI-boosting.

Tissue Parenchymal Cell Expression of B7-H1 Inhibits Infiltrating T Cell Expansion and Prevents Persistence of Graft-Versus-Host Disease

Abstract 2974Alloreactive donor CD8+ T cells facilitate engraftment and mediate graft versus leukemia (GVL) effects but also cause graft versus host disease (GVHD) in murine and human recipients after allogeneic hematopoietic cell transplantation (HCT). B7-H1 (PD-L1) expression by antigen-presenting cells has an important role in tolerizing activated T cells by binding to PD-1. We and others previously reported that disruption of binding between B7-H1 and PD-1 augments acute GVHD. Parenchymal cells do not usually express B7-H1 but can be induced by inflammatory cytokines (i.e. IFN-g) to express B7-H1. The role of B7-H1 expression by parenchymal tissue cells in regulating the expansion and persistence of donor CD8+ cells in tissues of mice with GVHD has not yet been evaluated. In the current studies, we evaluated the role of B7-H1 expression by GVHD target tissues in regulating donor CD8+ T cell function in 3 different experimental GVHD systems, using in vivo bioluminescent imaging (BLI), in vivo BrdU-labeling, and in vitro proliferation assays. The first system evaluated the role of B7-H1 expression in TBI-conditioned recipients. In these recipients, injected donor CD8+ T cells showed two waves of expansion that correlated with two phases of clinical GVHD. The first wave of donor CD8+ T cell expansion was associated with upregulated expression of B7-H1 in GVHD target tissues and only weak clinical GVHD. The second wave of donor CD8+ T cell expansion was associated with loss of B7-H1 expression, vigorous donor CD8+ T proliferation and expansion in the GVHD target tissues, and lethal GVHD. In a gain-of-function experiment, B7-H1 expression was induced in hepatocytes by hydrodynamic injection of B7-H1 cDNA during the second wave of T cell expansion in mice with GVHD; this subsequently decreased T cell expansion in the liver and ameliorated GVHD. The second system evaluated the role of B7-H1 expression in anti-CD3-conditioned recipients. In wild-type recipients, injected donor CD8+ T cells had only a single wave of expansion, and the mice had no signs of GVHD. B7-H1 expression by tissue cells (i.e. hepatocytes) was up-regulated, and the tissue infiltrating donor CD8+ T cells were anergic. In B7-H1−/− recipients, injected donor CD8+ T cells proliferated vigorously in GVHD target tissues and caused lethal GVHD.The third system evaluated the role of B7-H1 in unconditioned Rag-2−/− recipients after administration of blocking anti-B7-H1 and in the B7-H1−/−Rag-2−/− chimeras with B7-H1 sufficient Rag-2−/− bone marrow cells, in which B7-H1 deficiency was only in tissue parenchymal cells. Both blockade of B7-H1 and B7-H1 deficiency in parenchymal cells resulted in vigorous donor CD8+ T proliferation in GVHD target tissues and caused lethal GVHD. Taken together, these results show that expression of B7-H1 in GVHD target tissue parenchymal cells plays an important role in regulating the proliferation of infiltrating donor CD8+ T cells and preventing the persistence of GVHD. Our studies also indicate that TBI but not anti-CD3 conditioning can lead to loss of GVHD target tissue cell expression of B7-H1 and persistence of GVHD. Disclosures:No relevant conflicts of interest to declare.

Retransplantation From Haploidentical Donors After Graft Failure in Pediatric Patients Results in Low Transplant Related Mortality and Favourable Long Term Survival

Abstract 1956Graft failure is a rare but life-threatening complication after hematopoetic stem cell transplantation. Treatment comprises immunoablative reconditioning regimens and a second stem cell donation as soon as possible to minimize the time of pancytopenia and its sequelae. We report a cohort of 21 pediatric patients with acute leukemias (complete remission (CR)1-2 n= 7, active disease n=4; lymphatic n=7, myeloic n=4), myelodysplastic syndrome (n=2), immunodeficiencies (n=3), aplastic anemias (n=3), and hemoglobinopathies (n=2) who experienced graft failure (nonengraftment n=1; rejection n=20) after busulphan or melphalan based myeloablative transplantation from mismatched related donors (n=14) or matched unrelated or related donors (n=7). All patients were retransplanted with T cell depleted grafts (CD34 positive selection n=3 or CD3/CD19 depleted grafts n=18) from a haploidentical donor. Median time from diagnosis of graft rejection to second transplantation was 19 days. Reconditioning regimens consisted of irradiation (total lymphnode irradiation 7Gray (Gy) or TBI 2 Gy), fludarabine (120mg/m2) in combination with thiotepa (5mg/kg) or cyclophosphamide (60mg/kg body weight (bw)) and ATG/OKT3. A median number of 15×106/kg of body weight stem cells with 54×103/kg residual T cells were infused. Mofetilmycophenolat was given as Graft vs. Host Disease (GvHD) prophylaxis, if residual T cells exceeded 25 000/kg bw. Sustained engraftment was achieved in 20 out of 21 patients. One patient died before engraftment. Median time to absolute neutrophile counts above 500/μl was 9 (9–24) days. Independence from platelet substitution was reached at a median time of 10 (8–22) days. Only 1 patient developed GvHD °III, 10% developed GvHD °II, 33% of all patients developed GvHD °I, and 52% had no signs of GvHD. T cell recovery was delayed, however no lethal viral infection occurred. Severe organ toxicity was observed in 5 patients (bronchiolitis obliterans n=1, hemorrhagic cystitis n=2, leukencephalopathy n=2). Event free survival (EFS) of all patients at 5 years was 71%. 5 year EFS of patients with leukemias in complete remission was 83%. Patients with non-malignant diseases had 5 year EFS of 80%. Transplant related mortality at one year was 10%. Causes of death were: multi organ failure (n=1), fungal infection (n=1) and bronchiolitis obliterans organizing pneumonia, 4 patients with acute leukemias relapsed. None of the patients rejected the second graft. Thus, transplantation of stem cells from haploidentical donors after reconditioning is a realistic option to rescue patients with graft failure within a short time span. Graft rejection itself may have an antileukemic effect since patients with malignant diseases showed a favourable EFS. The use of a different donor in combination with irradiation based conditioning regimens may help to avoid a second rejection and results in a robust engraftment and low TRM. Disclosures:No relevant conflicts of interest to declare.

Graft T Cells Retard Stem Cell Derived Lymphoid Organ Reconstitution and Immune Function After Allogeneic Transplantation,

Abstract 4030Impaired immune function is a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Adoptively transferred donor T cells (TC) mediate graft-versus-host disease (GVHD), and make the use of pharmacological immunosuppression necessary, yet are believed to protect against infections post-HCT (pTX). In an MHC-matched, minor antigen mismatched mouse model, we studied the impact of allogeneic doTC on GVH-related damage of lymphoid organs, their recovery and immune function pTX. Lethally irradiated BALB.B mice received purified hematopoietic stem cells (HSC; cKit+Sca1+Thy1.1loLin−) +/− TC from C57BL/6 (B6) donors. Recipient lymphoid organs, marrow and livers were analyzed at multiple time points. Mice given HSC+TC promptly became full donor chimeras in all lineages and developed moderate GVHD. HSC recipients retained some residual host cells, and had no signs of GVHD. By tetramer analysis and ELISPOT assay, we showed that in pure HSC recipients early pTX residual host TC protected against murine cytomegalovirus infection. Once a nascent donor HSC-derived TC pool was established, these TC reacted robustly against the virus. In contrast, transferred mature allogeneic TC proliferated and expanded in HSC+TC recipients, but did not respond or protect against the virus. To better understand why MHC-matched mature donor TC did not react against this pathogen, even when derived from pre-immunized donors, we studied the lymphatic microenvironment, hypothesizing that only highly organized interactions between cellular and humoral immunity, antigen-presenting and innate immune cells provide full immune function. Comparing tissues of mice given pure HSC +/− TC we found that: (i) lethal radiation caused marked reduction in lymphoid organ cellularity at 2 wks pTX in all transplanted groups. While the cellularity increased rapidly in HSC recipients, severe hypocellularity persisted in HSC+TC recipients, was associated with significantly decreased organ size (lymph nodes [LN] and thymuses) and marked disruption of the histological architecture; (ii) TC phenotypes present in reconstituted mice comprised a mixture of naïve (CD62L+CD44−), central memory (CM; CD62L+CD44+) and effector memory (EM; CD62L−CD44+) cells in HSC recipients, but were largely EM and rarely naïve TC in HSC+TC recipients; (iii) at ∼3 wks pTX donor B cells (BC) dominated lymphoid tissues in HSC recipients, but were absent in HSC+ToTC recipients; (iv) NK cells regenerated promptly and steadily in HSC recipients, constituting up to 20% of all lymphoid cells in the liver at 3 wks pTX, while in HSC+TC recipients they incessantly decreased to near-complete absence; (v) in mesenteric (m) LN of HSC-, but not HSC+TC- recipients, CD4+CD8+CD3− TC were observed, derived from donor HSC. This latter population resembled immature thymic TC, suggesting that peripheral lymphoid organs participate in lymphoid reconstitution in adult recipients; (vi) at 2 wks pTX we noted only in HSC, but not HSC+ToTC recipients persisting and regenerating innate lymphoid cells (ILC). No ‘classical’ lymphoid tissue inducer cells, as described in embryogenesis of lymphoid organs, but cells with features of ILC were identified. Donor HSC-derived NKp46+RORgt+CD3− NK-like cells and IL-17A-secreting residual host CD4 TC were present, especially in livers and mLN. Cells that secreted IL-17A only and IL-17A+IFNγ were identified. IL-17A was not detected in recipients of HSC+ToTC, rather their donor TC were characterized by high IFNγ levels. Our studies show that, paradoxically, pure HSC grafts have advantages over TC-replete grafts with regard to immune function pTX. We hypothesize this superiority may be due to the presence of ILC that participate in the repair of lymphoid organs, and the whole spectrum of immune cells, including TC, BC, innate and immature cells emerging in HSC recipients. An intact microenvironment appears to be critical for optimal activation of TC to respond to pathogens. Moreover, our studies imply that donor TC cannot exert their protective immune function, when rapidly expanding alloreactive TC prevent the development of other lymphoid and innate immune cell, and disrupt the lymphoid tissue architecture. Controlling the negative effects of mature donor TC remains a challenge, and requires better understanding of the complexity of functional immune reconstitution pTX. Disclosures:No relevant conflicts of interest to declare.

T-Cell Depleted Allogeneic Hematopoietic Stem Cell Transplantation with Busulfan, Melphalan, and Fludarabine Conditioning Followed by Post Transplantation Donor Lymphocyte Infusions for Patients with Relapsed Multiple Myeloma and High-Risk Cytogenetics

Abstract 1992Allogeneic hematopoietic stem cell transplantation (allo HSCT) is a curative therapy for patients (pts) with multiple myeloma, but conventional allo HSCT has been associated with unacceptably high rates of mortality. Non-myeloablative allo HSCT has resulted in high rates of acute and chronic graft-versus-host disease (GvHD) and progression. We report results of a pilot study of 13 pts, using T-cell depleted allo HSCT (allo TCD HSCT) from HLA compatible (matched related = 8, matched unrelated = 3, and mismatched unrelated = 2) donors. All 13 pts had relapsed myeloma within 12 mos following auto HSCT, and 12/13 pts also had high-risk cytogenetics at diagnosis [t(4;14), t(14;16), del17p by FISH and/or del13q by karyotyping]. All pts achieved at least a partial response from preceding chemotherapy or second auto HSCT. Pts underwent allo TCD HSCT with busulfan (0.8mg/kg × 10 doses), melphalan (70mg/m2 × 2 days), fludarabine (25mg/m2 × 5 days) and rabbit ATG (2.5mg/kg × 2 days). T-cell depletion was performed by positive CD34 selection (Isolex) followed by rosetting with sheep erythrocytes, achieving < 103CD3+/kg for all grafts. All pts engrafted promptly (median d+11, range d+10 to +12). Pts were eligible to receive low doses of donor lymphocyte infusions (DLI) (5×10e5 – 1×10e6 CD3+/kg) no earlier than 5mos post allo HSCT.9/13 pts are alive and now 19–45months (mos) post TCD HSCT. Four pts are in complete remission (CR) at 19, 22, 33 and 39mos following allo TCD HSCT. Two of these pts reentered CR following DLI. Two pts had stable VGPR for 24 and 26mos before progression and reentered VGPR following treatment with Rev/Vel/Dex (RVD) + DLI; they are now 36 and 45mos post TCD HSCT. Two pts with refractory myeloma, who were transplanted with residual 16% and 10% plasma cells in marrow, achieved CR for 8 and 12mos post allo TCD HSCT before they developed progression. Following salvage chemotherapy + DLI, these pts are 20 and 21mos post transplant, respectively. One pt remained in CR for 9mos post TCD HSCT before progression with soft tissues plasmacytomas. With salvage radiation + chemotherapy, the pt is now 20mos post TCD HSCT. The 1 and 2 year probability estimates for overall survival and progression free survival with their 95% confidence intervals are: 1-yr OS 0.69 (95% CI: 0.48, 1.0); 2-yr OS 0.69 (95% CI: 0.48, 1.0); 1-yr PFS 0.46 (95% CI: 0.26, 0.83), 2-yr PFS 0.35 (95% CI: 0.15, 0.78).12/13 pts were without signs of GvHD, but one pt had possible superimposed gut GvHD following fulminant C diff colitis. Four pts died early post TCD HSCT (between 2–6 mos), due to oseltamivir-resistant H1N1 infection (N=1); respiratory failure secondary to infection of unknown etiology (N=1), status epilepticus (N=1), and acute cerebral hemorrhage (N=1).In summary, these results demonstrate that long-lasting disease control can be achieved with TCD HSCT in pts with multiply relapsed and refractory myeloma including those with high-risk cytogenetics. Administration of calculated, low dose donor lymphocyte infusions can induce complete remission without inducing GvHD. Pts who failed to respond to standard chemotherapy pretransplant responded to reuse of this therapy post TCD HSCT. Based on these results, we are performing a phase II clinical trial at Memorial Sloan-Kettering Cancer Center for pts with relapsed multiple myeloma following auto SCT who had high-risk cytogenetics at diagnosis or at relapse as well as for patients with high-risk cytogenetics in the upfront setting following preceding auto SCT. Disclosures:No relevant conflicts of interest to declare.

Inhibition of Histone Methylation Arrests Ongoing Graft-Versus-Host Diseases in Mice by Selectively Inducing Apoptosis of Alloreactive Effector T Cells

Abstract 820Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment option for patients with hematological malignancies. However, its success is limited by life-threatening graft-versus-host disease (GVHD). Novel approaches are needed to control GVHD. Recent studies have shown the importance of histone methylation in regulating the expression of genes associated with effector T cell differentiation and proliferation. Using several mouse models of allo-HSCT, we report that in vivo administration of the histone methylation inhibitor 3-Deazaneplanocin A (DZNep) arrested ongoing GVHD while preserving graft-versus-leukemia activity (GVL). To assess the therapeutic effect of pharmacologic modulation of histone methylation on GVHD, we administered DZNep to BALB/c mice receiving major histocompatibility-mismatched C57BL/6 mouse T cells 7 days after transplantation, in which GVHD had been fully established. Notably, injection of 12 doses of DZNep controlled the disease in these recipients, with approximately 80% of them surviving long-term without significant clinical signs of GVHD. We found that in vivo administration of DZNep caused selective apoptosis in alloantigen-activated T cells, but did not impair the generation of effector T cells that produced inflammatory cytokines (e.g., TNF-α, IFN-γ and IL-17) and cytotoxic molecules (e.g., granzyme B and Fas ligand). As a result, alloreactive T cells retained potent GVL activity, leading to improved overall survival of the recipients challenged by leukemic cells. These data suggest that DZNep-mediated inhibition of GVHD may be accounted for by reduced number of alloreactive effector T cells. In vitro culture assays showed that DZNep treatment induced apoptosis in T cells activated by anti-CD3/CD28 antibodies but not in naive T cells stimulated by IL-2 or IL-7. This effect was associated with DZNep’s ability to selectively reduce trimethylation of histone H3 lysine 27 (H3K27), deplete the histone methyltranferase Ezh2 that specifically catalyzes trimethylation of H3K27, and activate Ezh2-repressed pro-apoptotic gene Bim. Inactivation of Bim partially protected alloreactive T cells from DZNep-mediated apoptosis. Importantly, unlike DNA methylation inhibitors, inhibition of histone methylation by DZNep had no toxicities to hematopoietic cells or impairment on the reconstitution of hematopoiesis and thymopoiesis. Our findings indicate that modulation of histone methylation may have significant implications in the development of novel approaches to treat established GVHD and other T cell-mediated inflammatory disorders in a broad context. Disclosures:No relevant conflicts of interest to declare.