ALBERT

Description: Administration of the immunosuppressive drug cyclosporine A (CsA) following autologous stem cell transplantation paradoxically elicits a systemic autoimmune syndrome resembling graft-versus-host disease (GVHD). This syndrome, termed autologous GVHD, is associated with autoreactive CD8+ T cells that recognize major histocompatibility complex (MHC) class II determinants in association with a peptide from the invariant chain. To investigate the potential role of cytokines and chemokines in autologous GVHD, interleukin 2 (IL-2), IL-4, IL-10, interferon γ (IFN-γ), and macrophage inflammatory protein-1α (MIP-1α) gene expression in peripheral blood mononuclear cells (PBMCs) was determined in 36 patients treated with CsA following transplantation and correlated with the induction of cytolytic activity against autologous phytohemagglutinin-stimulated lymphocytes (PHA-blasts) and the breast cancer cell line (T47D). The determination of gene expression by real-time polymerase chain reaction (PCR) revealed that IL-10 mRNA levels by PBMCs in patients with autologous GVHD were 29-fold higher than in healthy individuals. IFN-γ (4-fold), IL-2 (3-fold), and MIP-1α (44-fold) mRNA levels were also increased in GVHD-induced patients compared with healthy individuals. The ability of PBMCs to lyse autologous PHA-blasts and T47D tumor cells exhibited an identical temporal relationship with expression of IL-10 and IFN-γ during autologous GVHD. Moreover, the susceptibility to autologous GVHD as assessed in 75 patients was significantly associated with the IL-10−1082 G/G polymorphic alleles, allelic variants in the promoter region that govern IL-10 production. These findings indicate that IL-10 may play an unexpected but critical role in autologous GVHD and could be utilized to enhance a graft-versus-tumor effect after transplantation. Interestingly, polymorphisms in the IL-10 promoter region may also explain differences in the susceptibility of patients to autologous GVHD induction.

Description: Syngeneic graft-vs-host disease (SGVHD) is a T cell dependent autoaggression syndrome induced by administering Cyclosporine following syngeneic bone marrow transplantation. The SGVHD autoreactive T cells recognize the MHC class II-invariant chain peptide complex (MHC class II-CLIP) and can be separated into functional subsets based on their differential dependence on the N- and C-terminal peptide flanking domains of CLIP. The present studies were undertaken to determine whether the N- or C-terminal flanking domain dependent subsets of CLIP reactive T cells reside within the CD4+CD25+ regulatory compartment. Multi-color flow cytometry was used to identify and isolate CD4+ (FITC) CD25+ (PE) T cells. Antigen-specific T cells within this compartment were identified with a soluble MHC class II-Ig chimeric construct (bioinylated, Cychrome avidin counterstaining) loaded with variants of CLIP containing the MHC class II binding domain and having either the N- or C-terminal flanking regions (N-CLIP, CLIP-C). Approximately 8.5% of the cells within the normal CD4+ lymphocyte population were CD25+. Both N-CLIP (1.1%) and CLIP-C (4.8%) reactive T cells coould be detected in the CD4+CD25+ population. Assessment of CD28, CTLA4, B7.1 and B7.2 mRNA expression levels by quantitative PCR directly ex vivo, revealed remarkable differences between the N-CLIP and CLIP-C specific CD4+CD25+ T cells. Although CD28 mRNA levels were comparable for both subsets, B7.2 and CTLA4 mRNA transcript levels were significantly increased (〉50 fold) in the CLIP-C+CD4+CD25+ T cells compared to the N-CLIP specific subset. On the other hand, levels of B7.1 mRNA were increased 〉10 fold in N-CLIP+CD4+CD25+ T cells. Additional studies assessing mRNA transcript levels for the regulatory transcription factor Foxp3, also revealed a disparity between the N-CLIP and C-CLIP specific subsets. mRNA transcript levels for Foxp3 were markedly increased (〉35 fold) in the CLIP-C dependent subset compared to the levels detected in N-CLIP+CD4+CD25+ T cells. Low levels of cytokine (IL-2, IL-4, interferon-γ) mRNA transcripts were detected in both subsets. Interestingly, intradermal immunization of normal animals with the peptides presented on dendritic cells increased mRNA transcript levels for type 1 cytokines in the N-CLIP reactive subset and type 2 cytokines in the CLIP-C dependent subset. Taken together, the results indicate that the CLIP-C antigen specific CD4+CD25+ cells have a profile consistent with regulatory T cells whereas the profile of the N-CLIP+CD4+CD25+ lymphocytes is more characteristic of activated T helper cells. The ability to identify and isolate regulatory T cells ex vivo and to modify their activity by immunization provides opportunities to both enhance and monitor the re-establishment of self-tolerance.

Description: Graft-versus-host disease (GVHD) is a serious, life-threatening complication that occurs following allogeneic (allo) bone marrow transplantation (BMT). The use of non-specific immunosuppression or T cell depletion has reduced the incidence of GVHD but at the expense of increased rates of infection and leukemic relapse. Modulation of the major costimulatory pathway (CD28/CTLA4:B7) involved in T cell activation and regulation may lead to specific immune tolerance in the absence of global non-specific immunosuppression. The identification of mRNA splice variants encoding for soluble forms of CD28, CTLA4 and GITR suggests that costimulation of T cells is complex and is not limited to cell-cell contact. The present studies examined the hypothesis that the onset of GVHD and the re-establishment of immune tolerance correlate with the expression levels of these costimulatory molecules. mRNA transcript levels for the soluble (s) and full-length (fl; cell surface associated) variants assessed by quantitative PCR, were temporally examined in peripheral blood lymphocytes (PBLs) from patients undergoing alloBMT (n=38) or autologous (auto) BMT (n=39) with the induction of autoGVHD by cyclosporin A treatment post-transplant. Levels of s and fl CD28 mRNA transcripts in PBLs were significantly increased (〉1.5 fold, P2.3-fold, P2.1-fold). sCTLA4 expression in patients with alloGVHD was significantly decreased than patients without alloGVHD. Interestingly, temporal analysis revealed that the levels for sCTLA4 paralleled the recovery from GVHD implicating an active process in the establishment of non-responsiveness. CD28, CTLA4 and GITR s and fl mRNA levels in CD4+CD25+ T regulatory (Treg) cells from allo and autoBMT patients were significantly increased (7-, 41- or 22-fold, P4 fold reduction of 3H-thymidine incorporation). However, pretreatment of the Treg subset with short interfering RNA (siRNA) to knockdown sCTLA4 gene (confirmed by quantitative PCR) significantly reduced the ability of these cells to suppress the response (minimal suppression was detected, 6%). In vitro siRNA studies also indicated that Treg cells with inhibited sCTLA4 expression were unable to suppress the response of IL-2-stimulated autoreactive CD8+ T cells. Taken together, the results indicate that increased expression of CTLA4 (soluble and cell-surface associated) and the “negative” signal delivered by this molecule to the T cell may regulate the development of GVHD and help to re-establish self tolerance after BMT. Defining the role of costimulation and the modulation of this pathway on immune recognition and regulation not only provides opportunities to enhance the re-establishment of tolerance but also may help to intensify anti-tumor immunotherapeutic strategies.

Description: BACKGROUND: Acute myeloid leukemia (AML) is an aggressive leukemia with 5-year overall survival of 20-25%. The major reason for treatment failure in AML is resistance to chemotherapy. Thus, there is an urgent need for identification of novel therapeutic agents for AML. Neoplastic cells, including AML, have dysfunctional redox regulation that results in increased reactive oxygen species (ROS). Accumulation of ROS leads to oxidation of free and incorporated nucleotides, leading to DNA damage and cell death. MTH1 is a nudix family hydrolase that sanitizes the oxidized nucleotide pool to prevent incorporation of these damaged bases in the DNA. MTH1 is thought to be non-essential for normal cells but crucial for neoplastic cells in order to avoid incorporation of oxidized dNTPs into DNA, thereby evading DNA damage and cell death. Whether MTH1 inhibitors have any activity against AML is not known. METHODS: Neoplastic myeloid cell lines HL-60, HEL, K562, KG1A, ML1, MV-4-11, SET2, and U937 were treated with varying concentrations of TH588 for a total of 48 hours. In experiments using the pan-caspase inhibitor Q-VD-OPh (Qvd), cells were pre-treated with 5µM Qvd for 1 hour followed by TH588. Cells were washed and stained with annexin, propidium iodide (PI), or MitoTracker (Life Technologies, Carlsbad, CA) for flow cytometry. To evaluate the potential impact of MTH1 inhibition on chemorefractory AML, HL-60/VCR cells were treated with vehicle control or TH588 in culture medium with or without vincristine (1µg/ml). Percentage apoptosis was calculated by normalizing to vehicle only control. With IRB approval, bone marrow aspirate samples were obtained from patients with untreated AML or healthy controls. Mononuclear cells were analyzed using colony-forming unit (CFU) assays. The total number of erythroid (CFU-E) and myeloid (CFU-G, CFU-GM) colonies containing ≥50 cells were read on day 14 and reported as percentage colonies compared to vehicle control. RESULTS: TH588 induced dose-dependent cell death in each of the neoplastic cell lines tested except HEL. In particular, treatment with TH588 resulted in a dose-dependent increase in the number of cells undergoing apoptosis as indicated by annexin V and/or PI staining (IC50 3.1-21.3µM, Figure 1). Pre-treatment with Qvd significantly inhibited TH588-induced cell death in all the cell lines studied except KG1A and SET2, suggesting a caspase-dependent mechanism of cell death. In further studies, cells treated with TH588 exhibited decreased MitoTracker staining; and Qvd pretreatment increased the number of MitoTrackerLow cells at the same time apoptotic cells decreased, suggesting that mitochondrial damage is upstream of caspase activation in TH588-induced apoptosis. Treatment with TH588 not only induced apoptosis in HL-60/VCR cells, but also facilitated further apoptosis in cells co-treated with vincristine and TH588 (Figure 2). Treatment with TH588 also diminished colony formation in a primary AML sample (IC50 6µM, Figure 3). Analysis of additional primary AML samples is ongoing. DISCUSSION: Our results show that the MTH1 inhibitor TH588 induces apoptosis in most neoplastic myeloid cells. MTH1 causes mitochondrial damage that, in turn, leads to caspase-dependent apoptosis in these cells. In HL-60/VCR cells representing chemorefractory phenotype, TH588 induces apoptosis as a single agent and resensitizes cells to vincristine. Moreover, TH588 significantly diminished colony formation in primary AML ex vivo. Further preclinical and possible clinical study of this class of agent appears warranted. Figure 1. Induction of cell death by MTH1 inhibitor TH588 in neoplastic myeloid cell lines. Figure 1. Induction of cell death by MTH1 inhibitor TH588 in neoplastic myeloid cell lines. Figure 2. TH588 induces apoptosis in HL-60/VCR cells and resensitizes cells to vincristine. Figure 2. TH588 induces apoptosis in HL-60/VCR cells and resensitizes cells to vincristine. Figure 3. TH588 significantly diminished colony formation in primary AML ex vivo indose-dependent manner. Figure 3. TH588 significantly diminished colony formation in primary AML ex vivo indose-dependent manner. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1009 Delayed immune reconstitution with increased risk of opportunistic infection is a major complication of HLA-haploidentical stem cell transplantation, especially in protocols employing extensive T cell depletion of the graft. Previous studies at our institution with high-dose, post-transplantation Cy (PT/Cy) have reported low rates of non-relapse mortality and serious opportunistic infections. Here we characterize immune reconstitution in fifty-three consecutive hematologic malignancies patients receiving nonmyeloablative conditioning, T cell-replete, HLA-haploidentical bone marrow transplantation (BMT), and graft versus host disease prophylaxis including PT/Cy. Patients with advanced hematologic malignancies (median age 51, range 14–71; 5 AML, 2 ALL, 4 MDS, 2 CML, 4 CLL, 1 CMML, 25 NHL, 7 Hodgkins, 3 mantle cell) received Cy 14.5 mg/kg/day IV on days −6 and −5, fludarabine 30 mg/m2/day IV on days −6 to −2, 200 cGy of TBI on day -1 and T cell replete bone marrow from donors with a median age of 44 (range 14–68). GVHD prophylaxis consisted of Cy (50 mg/kg/day) on days 3 and 4, mycophenolate mofetil for 30 days, and tacrolimus for 6 months. Grafts contained an infused median TNC/kg of 4.1 e8 (range 2.6–6.6 e8), CD3+/kg 3.6 e7 (range 1.7–6.7e7) and a CD34+/kg of 3.5e6 (range 1.4–7.0e6). Sustained engraftment of donor cells occurred in 86% of evaluable patients (44/51).The median times to neutrophil (〉500/μL) and platelet recovery (〉20,000/μL) were 17 days (range, 13–92 days) and 28 days (range, 13–580 days), respectively. Post-transplantation recovery of lymphocyte subsets is shown in Table 1 and Figure 1 and is notable for the following: 1) The median lymphocyte count at day 30 after transplantation is 〉180/ml and recovers to over 800/ml by day 60; 2) CD4+ T cell counts recover to a median 〉120/ml by day 60 and 〉220/ml by day 180 after transplantation; and 3) recovery of CD31+ recent thymic emigrants and CD45RA+ naïve T cells is delayed compared to recovery of memory T cells. T cell receptor spectratyping analysis on a subset of 10 patient/donor pairs chosen specifically for having no relapse/no GVHD (n=4), GVHD and no relapse (n=3), or late relapse (n=3) revealed that patients without relapse, GVHD, or recent viral infection had excellent reconstitution of the T cell repertoire to the level of the pre-transplant donor, as early as 6 months post-transplant (Figure 2). CMV specific T cell response using ELISPOT measured on a subset of 17 patients whose donors were reactive to CMV, revealed that donor-derived immunity to CMV returns by Day 60 in about 70% of patients (12/17) (Figure 3). In conclusion, immune reconstitution after non-myeloablative haploidentical T cell replete BMT with PT/Cy compares favorably with other reduced intensity conditioning alternative donor regimens and suggests that PT/Cy selectively preserves pathogen-specific memory T cells necessary to protect against infection. Further correlations of immune reconstitution with specific infectious and overall outcomes are being analyzed.Figure 1T-, B-, and NK-cell ReconstitutionFigure 1. T-, B-, and NK-cell ReconstitutionFigure 2T cell receptor spectratypingFigure 2. T cell receptor spectratypingFigure 3CMV-specific T cell frequencyFigure 3. CMV-specific T cell frequencyTable 1.Post-transplantation Lymphocyte Subset RecoveryMedian (cells/μL) (N)Interquartile range (cells/μL)ALCDonor1765 (46)1480–2100Recipient pre-BMT840 (45)425–1295Day 30184 (49)54–402Day 60820 (38)470–1260Day 180915 (34)670–1560Day 3653060 (22)820–2030CD3+CD4+CD45RA+ (naïve)Donor119 (33)82–189Recipient pre-BMT22 (34)4–38Day 300.33 (35)0.07–1Day 603 (29)1–9Day 18011 (23)5–31Day 36523 (13)13–92CD3+CD4+CD45RA−CCR7+ (central memory)Donor135 (33)95–158Recipient pre-BMT54 (34)11–79Day 302 (35)0.5–11Day 6034 (29)10–79Day 18061 (23)35–117Day 36589 (13)60–122CD3+CD4+CD45RA−CCR7− (effector memory)Donor187 (33)130–245Recipient pre-BMT87 (34)14–134Day 303 (35)1–18Day 6059 (29)14–122Day 180102 (23)43–179Day 365142 (12)64–204CD3+CD4+CD45RA+CD31+ (recent thymic emigrants)Donor61 (33)30–97Recipient pre-BMT6 (34)1–16Day 300.9 (35)0.03–0.4Day 601 (29)0.5–2Day 1804 (23)1–11Day 3657 (13)2–12CD3+CD4+Foxp3+Donor28 (33)23–35Recipient pre-BMT13 (34)6–24Day 301 (35)0.1–5Day 608 (29)4–16Day 18013 (23)7–25Day 36514 (13)7–17ALC, absolute lymphocyte count; WBC, white blood cell count; Treg, regulatory T cell Disclosures: Jones: Aldagen: Patents & Royalties.

Description: Key Points Agents that inhibit both complexes containing the mammalian target of rapamycin are particularly toxic to acute lymphocytic leukemia cells. This killing reflects engagement of a 4EBP1/c-MYC/PUMA axis downstream of mTORC1 and an NF-κB/EGR1/BIM axis downstream of mTORC2.

Description: Background Myelodysplatic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell malignancies, characterized by the ineffective hematopoiesis, and risk of progression to acute myeloid leukemia. Allogeneic stem cell transplant (SCT) remains the only potentially curative therapy, but toxicities limit its application in older adults. Vaccination strategies have been developed to modulate the immune system, ideally with less toxicity. We present results from a pilot study of single agent K562/GM-CSF whole cell vaccination in MDS patients (pts). Methods Poor-risk or transfusion-dependent MDS pts (n=5) were enrolled and received K562/GM-CSF whole cell vaccine (1 X 108 cells) every 3 weeks for 4 cycles followed by a booster vaccination 8 weeks later. Eligible pts could receive supportive care only for the 2 months preceding study entry, have no history of SCT, and not be on immunosuppressants. Blood, bone marrow, and skin biopsies were taken prior to vaccination, day 4 following first vaccine, and at 4 weeks after the booster vaccination. Diversity of the T cell compartment was assessed in the blood, marrow and skin using the TCRExpress Quantitative Analysis Kit (Biomed Immunotech, Tampa FL). Fragment length analysis was performed by the DNA Analysis Facility. In vitro stimulation studies for proliferation and cytokine production were performed using peripheral blood monocytes (PBMCs) for the pt with the best clinical response (〉4 years). Proliferation of banked PBMCs, collected prior to every vaccination and stimulated with the vaccine, was assessed using 3H-Thymidine. The cytokines generated were measured using a multiplexed bead based immunoassay (Human 17-plex bioplex pro-panel) (Biorad, Hercules CA). Results All pts tolerated the vaccinations well with localized skin reactions being common. Clinically, 2 pts normalized their blood counts and became transfusion independent following the immunotherapy. One responding pt (CMML) remained stable without need for medical intervention for 4 yrs and only recently showed progression. T cell spectratyping indicated oligocolonal populations of T cells in post-vaccination skin, blood and marrow samples. T cells infiltrating the skin were tracked to the marrow. Interestingly, the best clinical responder demonstrated the most restricted skewed repertoire with a significant number of oligoclonal T cells tracking from skin to marrow (n=5). The marrow also had infiltrates of oligoclonal T cells not detected in the post-vaccination skin. Further, this skewed repertoire was absent when the pt relapsed. Proliferation assays measuring this pt’s T cell cytokine production in response to the vaccine cells in vitro, showed increased levels of IL-2, IL-13 and IL-5 that were suppressed or not produced by the time of the 4th vaccination. Inversely, IL-6, IL-10, IL-17, IL-1beta, MIP-1beta and TNF alpha levels increased throughout. The expected proliferative “boost” was seen with the initiation of the booster vaccine series at the time of progression, and co-culture of the pt’s lymphocytes with the vaccine cells suppressed the ability of the vaccine cells to produce GM-CSF in vitro. The ability to suppress GM-CSF production decreased during therapy and the pt’s lymphocytes had no effect on GM-CSF production by the vaccine at the end of the immunotherapy. Conclusions All pts showed T cell skewing by spectratyping analysis, suggesting that each had a change in T cell proliferation patterns in response to the vaccine. One pt had a significant clinical response and the most specific T cell response by spectratyping to the original vaccine, followed by the absence of these cells in the marrow at the time of progression. This suggests that an immune response may have stabilized his disease and progression was associated with loss of this T cell population. Proliferation studies suggest that the lymphocytes recognized the vaccine. Lastly, GM-CSF levels produced by the vaccine were decreased during the vaccination cycles suggesting that the pt’s lymphocytes and/or tumor had a suppressive effect on the vaccine cells. It is unclear if the GM-CSF suppression was essential, detrimental, or unrelated to the pt’s clinical response. Further study of the T cells patterns in these pts may elucidate details of the immune response that are integral to clinical responses. Disclosures: No relevant conflicts of interest to declare.