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Few published studies characterize early lymphocyte recovery after intensive chemotherapy for acute myelogenous leukemia (AML). To test the hypothesis that lymphocyte recovery mirrors ontogeny, we characterized early lymphocyte recovery in 20 consecutive patients undergoing induction timed sequential chemotherapy for newly diagnosed AML. Recovering T lymphocytes were predominantly CD4+ and included a greatly expanded population of CD3+CD4+CD25+Foxp3+ T cells. Recovering CD3+CD4+CD25+Foxp3+ T cells were phenotypically activated regulatory T cells and showed suppressive activity on cytokine production in a mixed lymphocyte reaction. Despite an initial burst of thymopoiesis, most recovering regulatory T cells were peripherally derived. Furthermore, regulatory T cells showed marked oligoclonal skewing, suggesting that their peripheral expansion was antigen-driven. Overall, lymphocyte recovery after chemotherapy differs from ontogeny, specifically identifying a peripherally expanded oligoclonal population of activated regulatory T lymphocytes. These differences suggest a stereotyped immunologic recovery shared by patients with newly diagnosed AML after induction timed sequential chemotherapy. Further insight into this oligoclonal regulatory T-cell population will be fundamental toward developing effective immunomodulatory techniques to improve survival for patients with AML.

Graft-versus-host disease (GVHD) is characterized by an impairment of mechanisms that underlie the development of immunologic tolerance. Although the cytokine storm associated with GVHD leads to expression of cell surface markers on both effector and regulatory T cells, regulatory CD4+ T cells that play an instrumental role in the maintenance of tolerance appear to uniquely express the Foxp3 transcriptional repressor. Foxp3 mRNA expression was significantly decreased in peripheral blood mononuclear cells from patients with either allogeneic GVHD or autologous GVHD compared with patients without GVHD. Expression of Foxp3 negatively correlated with the severity of GVHD but positively correlated with recent thymic emigrants. The results suggest that defective thymic function contributes to the impaired reconstitution of immune regulatory mechanisms following transplantation. The decrease in regulatory mechanisms after transplantation appears to provide an environment permissive to the development of GVHD. (Blood. 2004;104:2187-2193)

Administration of cyclosporine A (CsA) after autologous stem cell transplantation elicits an autoimmune syndrome with pathology similar to graft-versus-host disease (GVHD). This syndrome, termed autologous GVHD, is associated with the appearance of autoreactive T cells directed at major histocompatibility class (MHC) class II antigens. In the rat model of autologous GVHD, clonal analysis reveals that the effector T cells are highly conserved and recognize a peptide from the invariant chain peptide presented by MHC class II. Although human autologous GVHD effector T cells share a similar phenotypic specificity, clonality of the response in humans has not been determined. To examine the human effector T-cell response, the T-cell repertoire of peripheral blood lymphocytes was assessed by complementarity-determining region 3 (CDR3) size distribution analysis and T-cell clonotype analysis in 26 patients treated with CsA after transplantation. Autologous GVHD developed in 3 of 4 patients with human leukocyte antigen (HLA)-DRB1*0701, and clonal expansions of β-chain variable region (BV)16+ T cells were shared. Clonal expansions within BV15+ and BV22+ T cells were also detected in 4 of 6 patients with HLA-DRB1*1501 and in 3 of 4 patients with HLA-DRB1*0401, respectively. Sequencing of BV16 cDNA for which the CDR3 size pattern exhibited apparent clone predominance revealed an identical CDR3 peptide sequence in 2 different patients, one with HLA-DRB1*0701 and the other with HLA-DRB1*1502. These findings indicate that the discrete antigen-driven expansion of T cells is involved in autologous GVHD.

Administration of the immunosuppressive drug cyclosporine after autologous bone marrow transplantation induces a systemic autoimmune syndrome resembling graft-versus-host disease (GVHD). This syndrome termed autologous GVHD has significant antitumor activity. Associated with autologous GVHD is the development of T lymphocytes that recognize major histocompatibility complex (MHC) class II determinants, including self. The present studies attempted to characterize and define the molecular specificity of the effector T lymphocytes in autologous GVHD induced in patients with metastatic breast cancer. The results suggest that the effector cells associated with human autologous GVHD are CD8+ T lymphocytes expressing the α/β T-cell receptor. Additional studies show that the effector T cells recognize MHC class II antigens in association with a peptide from the invariant chain (CLIP). Pretreatment of autologous lymphoblast target cells with anti-CLIP antibody completely blocked lysis mediated by autologous GVHD effector T cells. On the other hand, force loading this peptide markedly enhanced the susceptibility of the target cells to recognition by the autoreactive T cells. The recognition of the MHC class II CLIP complex may account for the novel specificity of the effector T cells associated with human autologous GVHD. Moreover, identification of the target peptide may allow for the development of novel immunotherapeutic strategies to enhance the antitumor efficacy of autologous GVHD.

The mammalian target of rapamycin (mTOR) plays crucial roles in proliferative and antiapoptotic signaling in lymphoid malignancies. Rapamycin analogs, which are allosteric mTOR complex 1 (mTORC1) inhibitors, are active in mantle cell lymphoma and other lymphoid neoplasms, but responses are usually partial and short-lived. In the present study we compared the effects of rapamycin with the dual mTORC1/mTORC2 inhibitor OSI-027 in cell lines and clinical samples representing divers lymphoid malignancies. In contrast to rapamycin, OSI-027 markedly diminished proliferation and induced apoptosis in a variety of lymphoid cell lines and clinical samples, including specimens of B-cell acute lymphocytic leukemia (ALL), mantle cell lymphoma, marginal zone lymphoma and Sezary syndrome. Additional analysis demonstrated that OSI-027–induced apoptosis depended on transcriptional activation of the PUMA and BIM genes. Overexpression of Bcl-2, which neutralizes Puma and Bim, or loss of procaspase 9 diminished OSI-027–induced apoptosis in vitro. Moreover, OSI-027 inhibited phosphorylation of mTORC1 and mTORC2 substrates, up-regulated Puma, and induced regressions in Jeko xenografts. Collectively, these results not only identify a pathway that is critical for the cytotoxicity of dual mTORC1/mTORC2 inhibitors, but also suggest that simultaneously targeting mTORC1 and mTORC2 might be an effective anti-lymphoma strategy in vivo.

Graft-versus-host disease (GVHD) continues to be a major complication after allogeneic bone marrow transplantation, especially with the increasing use of unrelated and mismatched donors. Recently there has been renewed scientific interest in GVHD because of the increasing appreciation of the complexity of the immune responses seen in GVHD. Two basic aspects of the immune response in GVHD, the immunologic target and the effector mechanisms, are now more completely understood. First, the target of the immune response in GVHD has long been felt to be histocompatibility antigens possessed by the host, but not the donor. Recently, recognition of self antigens in GVHD has been documented, showing that GVHD is more complex than simple alloreactivity. Second, the effector mechanism in GVHD was initially felt to be direct cytotoxicity by alloreactive T cells. It is now recognized that cytokines play a central role in mediating many of the clinical and experimental manifestations of GVHD. The development in these two areas will be reviewed and the implications for clinical transplantation discussed.

Cutaneous graft-versus-host disease (GVHD) has been reported after administration of cyclosporine (CSP) after autologous bone marrow transplantation (ABMT) with unpurged marrow in patients with lymphoma. To determine whether GVHD can be induced after ABMT with chemopurged marrow in acute myeloid leukemia (AML), we administered intravenous CSP for 28 days (beginning on the day of ABMT) to 19 patients with AML (12 in first remission [CR1], six in CR2, and one in CR3) who received busulfan (16 mg/kg) and cyclophosphamide (200 mg/kg) and ABMT with 4- hydroperoxycyclophosphamide (4HC)-treated marrow. In this dose- escalation trial, CSP daily doses were 1 mg/kg in seven patients, 2.5 mg/kg in eight patients, or 3.75 mg/kg in four patients. Skin biopsies were obtained weekly after ABMT or on appearance of rash and were graded for GVH changes. Overall, 15 of 19 patients (79%) had cutaneous histopathologic grade 2 GVHD at a median of 33 days (range, 14 to 49) after ABMT; in 10, cutaneous manifestations were present at time of positive biopsy. The frequency, time to onset, and duration of GVHD were similar among the three CSP dosage groups. No patients had hepatic or gastrointestinal dysfunction attributable to GVHD or required specific therapy for GVHD. Positive biopsies for GVHD were seen in seven of eight patients who received full-course, full-dose CSP and 8 of 11 patients who had CSP discontinued or dosage reduced because of renal insufficiency. Three patients (one with positive biopsy) died with ABMT-related complications. Seven patients (four CR1, three CR2) relapsed with AML at a median of 411 days (range, 178 to 549) after ABMT; six of seven had positive biopsies for cutaneous GVHD. Nine patients (seven CR1, one CR2, and one CR3) are alive without relapse at a median of 501+ days (range, 252+ to 811+) after ABMT; eight of nine had cutaneous GVHD. Short-course CSP can induce autologous GVHD in recipients of chemopurged marrow autografts for AML, but randomized prospective trials are needed to determine whether this immunologic reaction is associated with alterations in leukemic relapse rate and disease-free survival after ABMT in AML.

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.

The mammalian target of rapamycin (mTOR), a kinase that regulates proliferation and apoptosis, has been extensively evaluated as a therapeutic target in hematologic malignancies. Rapamycin analogues, which partially inhibit mTOR complex 1 (mTORC1), showed limited anti-tumor activity due to feedback mechanisms involving mTORC2 and incomplete inhibition of mTORC1. Thus, attention has turned to agents targeting both mTOR complexes by binding the mTOR kinase domain. The purpose of this study was to delineate the mechanisms of mTOR dual inhibitor induced apoptosis in human neoplastic lymphoid cells in vitro. MTS assays and propidium iodide staining followed by flow cytometry for subdiploid populations demonstrated that OSI-027 and MLN0128 inhibited cell proliferation and induced apoptosis in a wide range of lymphoid cell lines including Jurkat, Nalm-6, Molt-4, and SeAX. Raptor and Rictor knockdown in Jurkat and Nalm-6 increased cell death, suggesting both mTOR complexes play a role in apoptosis. 4EBP1 phosphorylation was inhibited by mTOR dual inhibitors, but not by rapamycin. Expression of 4EBP1 T37A/T46A and 4EBP1 T37A/T46A/S65A/T70A, which mimic dephosphorylated 4EBP1, increased Puma mRNA and protein levels as well as apoptosis. Moreover, 4EBP1 knockdown abrogated mTOR dual inhibitor induced Puma upregulation and cell death, further supporting the role of 4EBP1 dephosphorylation in mTORC1 dependent apoptosis. In accord with the known dependence of c-Myc translation on the eIF4E/eIF4G complex, we also observed c-Myc downregulation after treatment with OSI-027, MLN0128 and 4EGI-1, but not rapamycin. Puma induction mirrored c-Myc downregulation under a variety of conditions, including expression of nonphosphorylatable 4EBP1 in parental Jurkat cells or wt 4EBP1 in 4EBP1 deficient cells. Furthermore, c-Myc knockdown induced Puma mRNA and protein as well as increased apoptosis. Collectively, these results support a model in which mTORC1 inhibition, acting through 4EBP1, induces Puma upregulation and apoptosis through c-Myc downregulation. In order to assess the parallel mTORC2-dependent Bim-mediated apoptotic mechanism, we utilized reporter assays and RNAseq experiments. OSI-027-induced Bim promoter activity decreased markedly when the nucleotides -29 to -18 were removed, suggesting that this response element is critical for OSI-027-induced promoter activation. In silico analysis identified eight transcription factors, including SP1, Egr-1, and Myb, that potentially bind this 12-bp region. In RNAseq experiments, we detected a 9-fold increase in Egr-1. Egr-1 upregulation was confirmed by qRT-PCR and immunoblotting after dual inhibitors treatment or Rictor knockdown. Moreover, dominant negative Egr-1 or Egr-1 knockdown diminished dual inhibitor-induced Bim promoter activation and Bim upregulation. Chromatin immunoprecipitation assays demonstrated that OSI-027 enhances binding of Egr-1 to a region of the Bim promoter including bp -29 to -18, further confirming that Egr-1 functions as a direct transcriptional activator for Bim upon mTOR dual inhibitor treatment. NFκB is a known transcription factor for Egr-1 and we observed increased p65 in the nucleus and increased NFκB transcriptional activity after dual inhibitor treatment. Overexpression of S32A/S36A IκB impaired the ability of dual inhibitors to induce NFκB transcriptional activation, Egr-1 mRNA and protein, Bim promoter activation and Bim mRNA and protein upregulation. Collectively, these results suggest that mTORC2 inhibition induces Bim upregulation and apoptosis through NFκB and Egr-1 transactivation. When fresh clinical ALL isolates were exposed to OSI-027 or MLN0128 ex vivo, inhibition of 4EBP1 phosphorylation along with upregulation of Egr-1 and Bim and/or c-Myc downregulation accompanied by Puma induction occurred, indicating that the pathways identified in ALL cell lines can also potentially be engaged in clinical ALLs. These observations not only provide new insight into the survival roles of mTOR in lymphoid malignancies, but also identify alterations that potentially modulate the action of mTOR dual inhibitors. Disclosures No relevant conflicts of interest to declare.

MLN4924, a novel chemotherapeutic agent designed to inhibit the Nedd8 Activating Enzyme (NAE), has shown promise in early clinical testing in relapsed and refractory Acute Myeloid Leukemia (AML). NAE inhibition prevents transfer of the Nedd8 protein to Cullin Ring Ligases (CRLs), leading to their inactivation and subsequent accumulation of the protein substrates they normally ubiquitinate and target for degradation by the proteasome. In malignant cells treated with MLN4924, various CRL substrates accumulate and induce a cytotoxic effect. Importantly, the substrates responsible for these effects vary among different malignancies depending on basal gene expression patterns. In order to shed light on the mechanism of MLN4924 anti-leukemic effects and identify potential mechanisms of resistance, the effects of MLN4924 on AML cell lines and primary AML samples were examined in vitro and in vivo. These studies identified c-Myc as the critical substrate governing the cytotoxic effect. In particular, we observed c-Myc transactivation of the PMAIP1 gene, leading to expression of pro-apoptotic protein Noxa, which functions to neutralize Mcl-1, activate Bak and Bax to allow release of cytochrome c from the outer mitochondrial membrane, and induce apoptosis. Importantly, MLN4924-induced Noxa upregulation was prevented by c-Myc shRNA; and MLN4924-induced killing was markedly diminished by Noxa siRNA. In further experiments, a synergistic cytotoxic effect was seen when MLN4924 was applied to AML cell lines in combination with agents that inhibit anti-apoptotic proteins other than Mcl-1, such as BH3 mimetics ABT-263 (inhibits Bcl-2, Bcl-xL, and Bcl-2) and ABT-199 (inhibits Bcl-2). Because expression of anti-apoptotic proteins within leukemia samples and between different AMLs is variable, and Mcl-1 upregulation often contributes to drug resistance and relapse in AML, this combination may be advantageous in several clinical settings. To follow up on the effects we observed with MLN4924 alone and in combination with BH3 mimetics, we have designed a multi-parameter flow cytometry based assay to assess response to these agents among hematopoietic stem cells (HSCs) and progenitor populations in primary leukemia bone marrow isolates treated ex vivo. Pilot studies conducted in mononuclear cell fractions containing normal HSCs and hematopoietic progenitors isolated from umbilical cord blood did not undergo apoptosis in response to MLN4924. Ongoing studies aim to specifically assess whether MLN4924 alone or in combination with other anti-leukemic agents induce apoptosis or alter expression of CD47 (an anti-phagocytic protein frequently upregulated in AML) in HSCs and hematopoietic progenitor cell populations within primary AML bone marrow isolates. As our understanding of how HSCs and hematopoietic progenitors contribute to AML initiation and progression advances, the need to target various populations will emerge and may vary depending on disease stage. It will be critical to ascertain whether HSCs and other populations are sensitive or resistant to agents such as MLN4924 and others in order to formulate and apply efficient targeted therapies. Disclosures No relevant conflicts of interest to declare.

Regulatory T (Treg) cells can suppress a wide variety of immune responses, including antitumor and alloimmune responses. The mechanisms by which Treg cells mediate their suppressive effects depend on the context of their activation. We previously reported that granzyme B is important for Treg cell–mediated suppression of antitumor immune responses. We therefore hypothesized that granzyme B may likewise be important for suppression of graft-versus-host disease (GVHD). We found that allogeneic mismatch induces the expression of granzyme B in mixed lymphocyte reactions and in a model of graft-versus-host disease (GVHD). However, wild-type and granzyme B–deficient Treg cells were equally able to suppress effector T (Teff) cell proliferation driven by multiple stimuli, including allogeneicantigen-presenting cells. Surprisingly, adoptive transfer of granzyme B–deficient Treg cells prevented GVHD lethality, suppressed serum cytokine production in vivo, and prevented target organ damage. These data contrast strikingly with our previous study, which demonstrated that granzyme B plays a nonredundant role in Treg cell–mediated suppression of antitumor responses. Taken together, these findings suggest that targeting specific Treg cell–suppressive mechanisms, such as granzyme B, may be therapeutically beneficial for segregating GVHD and graft-versus-tumor immune responses.

Failure of the CD4+CD25+Foxp3+ regulatory T-cell compartment to control immune reactivity to self-antigens leads to the development of chronic graft-versus-host disease largely mediated by donor-derived helper T cells that secrete pro-inflammatory cytokines. Graft-versus-host disease (GVHD) remains

Comment on Rezvani et al, page [1291][1] The relative content of Foxp3+ regulatory T cells in the donor marrow inoculum correlates with the risk of developing graft-versus-host disease after HLA-matched allogeneic stem cell transplantation. Allogeneic stem cell transplantation has often proven a

Comment on Tivol et al, page [4885][1] The immunologic response to foreign alloantigens in graft-versus-host disease can lead to the breakdown of self-tolerance precipitating autoimmunity. G raft-versus-host disease (GVHD) is a major life-threatening complication of allogeneic bone marrow

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.

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.

OBJECTIVE: Investigate the contribution of PIG-A mutations to clonal expansion in paroxysmal nocturnal hemoglobinuria (PNH). INTRODUCTION: Whether PNH cells are inherently less susceptible to apoptosis remains controversial. Studies using PNH mouse models and lymphoid cell lines have failed to find a difference in apoptosis, but reports using K562 cells have found a relative resistance. Studies using primary CD34+ cells from PNH patients have also found a relative resistance of PNH cells, but not in comparison to CD34+ cells from normal controls. We designed experiments 1) to control for genetic heterogeneity among PNH and control cell lines, 2) to address the potential methodological issues associated with studying lymphocytes, which are rarely affected in PNH, and 3) address inconsistency comparing normal and PNH progenitors that have experienced different in vivo environments. METHODS: GPI-anchored protein (GPI-AP) positive and negative primary CD34+ hematopoietic progenitors from PNH patients were assayed for annexin V positivity by flow cytometry in a cell-mediated killing assay using autologous effectors from PNH patients or allogeneic effectors from healthy controls. To specifically assess the role of the PIG-A mutation in the development of clonal dominance and address confounders of secondary mutation and differential immune selection in vivo, we established an inducible PIG-A CD34+ myeloid cell line, TF-1. Using a doxycycline-inducible wild type PIG-A tet-SUPER transgene expression system, in which GPI-AP− and GPI-AP+ cells are isogenic, we assessed apoptosis resistance and clonal expansion in response to various pro-apoptotic stimuli. Apoptosis resistance was assessed after exposure to allogeneic effectors, NK92 effectors, TNF-α, and γ-irradiation. Apoptosis was measured by annexin V/PI staining (effector experiments) or caspase 3/7 activity (TNF-α and γ-irradiation experiments). Blocking experiments of NK92-mediated killing utilized mAb to ULBP1 and ULBP2, as per Hanaoka et al. Clonal competition experiments tracked wild type and PIG-A mutant TF-1 cells using flow cytometry after exposure to TNF-α as a surrogate for immune attack. RESULTS: In PNH patients, GPI-AP− CD34+ hematopoietic progenitors were less susceptible than GPI-AP+ CD34+ precursors to autologous (8% versus 49%, p CONCLUSIONS: PIG-A mutations contribute to the clonal expansion in PNH by conferring a survival advantage to hematopoietic progenitors under pro-apoptotic stresses. Lack of GPI-anchored ULBP1 and ULBP2 expression contributes to the apoptosis resistance in a cell-mediated killing model but is not solely responsible for the apoptosis resistance. The global apoptosis resistance translated into expansion of the PNH clone in an in vitro model of immune attack.

Recovery of immune function after initial treatment of acute myeloid leukemia (AML) is critical, not only for protection against infections but also for surveillance against recurrent disease. A better understanding of the nature of lymphocyte recovery following induction and consolidation therapy for AML could guide the design of immunotherapy strategies aimed at boosting the anti-leukemia activity of a reconstituted immune system. Prior studies examining thymic T cell production following bone marrow transplantation (BMT) have found varying levels of thymic output post-transplant, as measured by T cell Receptor Excision Circle (TREC) levels in the peripheral blood of adult patients. Of note, relapse of chronic myeloid leukemia (CML) following BMT is correlated with decreased levels of TREC positive T cells. In order to characterize immune reconstitution in AML, we studied 26 patients after induction or consolidation time sequential chemotherapy. Their median age was 52 (range 23–69). Thirteen patients received cytarabine, daunorubicin, and etoposide (AcDVP-16) induction therapy, 3 patients received cytarabine, daunorubicin, and cytarabine (AcDAc) consolidation therapy and 10 patients received flavopiridol, cytarabine, and mitoxantrone (FLAM) either as induction or consolidation therapy. Peripheral blood samples were collected approximately every other day for 3–5 time points after each patient’s white blood cell count exceeded 200 cells/cubic mm on three consecutive days. Among the four patients evaluated to date, flow cytometry results show that a majority of cells seen early in immune reconstitution are CD3+ lymphocytes (range 69–97%). Subset analyses on 3 of these 4 patients have shown CD4:CD8 ratios ranging from 3:1 to 4:1, while the fourth patient exhibited an inverse of this ratio at 1:5. In addition, CD25+FOXP3+ T cells represented a median of 5.1% (range 2.5–12.3%) of the CD3+ T cells. Since T cells represented the abundance of cells in the peripheral blood during early bone marrow recovery, we then assessed whether these cells represented recent thymic emigrants or naïve T cells by examining TRECs using real time PCR (RT-PCR). TRECs were present in 24 of the 26 patients with levels ranging between 100 and 100,000 copies per 100,000 cells. Furthermore, 4 control samples from normal volunteers (ages 37–43) revealed the absence of TREC positive cells. Further analyses of these time points and correlations between TREC levels and clinical responses are ongoing.

For patients with recurrent and refractory Hodgkin Disease (HD), the major risk after standard treatment with high-dose chemotherapy and autologous hematopoietic stem cell rescue (ASCR) is relapse. In the setting of allogeneic SCT, the graft-vs.-lymphoma effect is offset by high transplant-related mortality. The Children’s Oncology Group has therefore undertaken a study of immunotherapy to induce autologous graft-vs.-host disease (auto GVHD) in an effort to reduce the relapse rate without excessive morbidity and mortality. The objectives of part I of the study were to determine the toxicity and feasibility of inducing auto GVHD with cyclosporine, interferon-γ , and IL-2 after high-dose BEAM. Twenty-four patients with biopsy-confirmed recurrent/refractory HD were enrolled at 13 institutions. One patient was inevaluable because of a protocol violation. At initial diagnosis, 5 were stage IV, 8 stage III, 7 stage II, 1 stage I, and 3 unknown; 16 had B symptoms, and 15 had bulky disease. All patients had failed intensive multi-agent chemotherapy, and most had received prior radiation therapy. At relapse, 3 patients had B symptoms and 2 had bulky disease, and 8 had extranodal disease. All patients received full doses of the preparative regimen, with the expected reversible complications of febrile neutropenia, pancytopenia, nausea, vomiting, anorexia, mucositis, and electrolyte disturbances. In addition to fever, fatigue, and cytopenias, two patients developed pneumonitis after receiving immunotherapy, one of whom died of respiratory failure 6 wks after study entry. Of note, this patient received only 2 doses of IL-2 before developing pneumonitis. Bronchoalveolar lavage failed to demonstrate an etiology, but a culture obtained by open lung biopsy was positive for staphylococcus epidermidis. One patient developed a rash and one patient developed liver function abnormalities during the immunotherapy consistent with auto GVHD. Toxicities Toxicity RT/BEAM/ASCR Immunotherapy n = 16 n = 15 Febrile neutropenia 11 (69%) 4 (27%) Nausea/vomiting/anorexia 10 (63%) 3 (20%) Mucositis 7 (44%) 2 (13%) Metabolic/lab 8 12 Pneumonitis 0 2 (13%) Fever 0 2 (13%) Fatigue 0 2 (13%) Hypotension 1 (6%) 0 Blood was requested at weekly intervals during immunotherapy administration to test for autoreactivity in mixed lymphocyte cultures and by cytokine assays with autologous stimulator cells. The sample submission rate was 90%. In 11 of 14 patients, there was significant in vitro lymphocyte autoreactivity. We conclude that this immunotherapy regimen is tolerable and induces autoreactivity in a sufficient proportion of patients to warrant proceeding with part II of the study. Part II will test the efficacy of immunotherapy by randomizing patients with chemosensitive recurrent/refractory HD to receive immunotherapy or not after BEAM and ASCR.

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.

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.

Chronic graft-v-host disease (GVHD) is the most frequent late complication of allogeneic bone marrow (BM) transplant. To test different treatments, we used a rat model of chronic GVHD which clinically, histologically, and immunologically resembles the disease occurring after human marrow transplant. The following treatments were administered: azathioprine 50 mg/kg/day plus prednisone 1 mg/kg/every other day; Cyclosporine (CsA) 30 mg/kg/day; CsA 30 mg/kg/day plus prednisone 1 mg/kg/every other day; thalidomide 50 mg/kg/day; thalidomide 10 mg/kg/day; CsA 10 mg/kg/day; and thalidomide 10 mg/kg/day plus CsA 10 mg/kg/day. All drugs were administered by gavage. Half of the animals (six of 12) treated with azathioprine plus prednisone every other day responded to treatment. The majority of animals (seven of 12) treated with CsA died early. The addition of prednisone every other day did not improve survival. The surviving animals treated with CsA (with or without prednisone) responded to treatment. Half of the animals treated with each of the above regimens had recurrent or progressive disease after therapy was discontinued. Thalidomide treatment was successful in the majority of animals (16 of 18). The addition of low-dose CsA to low-dose thalidomide resulted in a faster rate of response (12.1 +/- 1.8 v 28.2 +/- 1.6 days). We conclude that both thalidomide and thalidomide plus CsA appear promising in this model as treatment for chronic GVHD.

Lethally irradiated LouM rats reconstituted with syngeneic bone marrow and then treated with cyclosporine (CsA) for 40 consecutive days following transplant developed a graft-v-host disease (GVHD)-like syndrome after CsA cessation. This model of GVHD was used to define and characterize a graft-v-tumor (GVT) effect against a syngeneic plasmacytoma CRL1662 cell line which expresses class II major histocompatibility (MHC) antigen (Ia). Nylon wool-nonadherent spleen cells from animals who developed syngeneic GVHD were capable of significant lysis against chromium-labeled tumor target cells in a four- hour chromium released cell mediated lympholysis assay; maximum lysis occurred five days following cessation of CsA when clinical signs first appeared. Cytolytic activity declined to baseline as GVHD symptoms resolved. Fractionation of splenocytes into lymphocyte subsets demonstrated that cytolytic lymphocytes (CTLs) of the OX8 phenotype (non-helper T) were capable of significant lysis against tumor target cells. Lysis of tumor cells was blocked by preincubation with monoclonal antibodies (MoAb) specific for the rat anti-class II MHC antigen but not with MoAb against class I. Incubation of tumor cells with gamma-interferon increased expression of tumor class II MHC antigens and significantly increased their susceptibility to lysis by nylon wool-nonadherent splenocytes from animals with syngeneic GVHD. These studies have demonstrated an in vitro GVT of syngeneic GVHD against an Ia-bearing tumor; the effector cell is a CTL of the OX8 phenotype specific for the class II MHC antigen.

Cyclosporin-A (CsA) was given to 22 patients who received allogeneic bone marrow transplants as therapy for aplastic anemia and hematologic malignancies. The drug was given daily for 180 days starting with the day of marrow infusion. Engraftment was not impaired and myelotoxicity was not observed. Cutaneous graft-versus-host disease (GVHD) developed in five patients and all either spontaneously resolved or promptly responded to therapy with steroids. Five patients developed systemic GVHD and all responded to therapy with steroids, but only two survived. Interstitial pneumonia was seen in six patients and was fatal in all of them. Liver function abnormalities were seen in 14 patients but could not positively be correlated with CsA administration. Renal function abnormalities were seen in 17 patients. Amphotericin-B therapy contributed significantly to the renal failure. Serum levels of CsA, measured by radioimmunoassay, could not be correlated with the presence of liver or renal function abnormalities. Overall survival so far has been 50.0%. Second malignancies were not observed, but one patient relapsed with leukemia at 343 days.

Cyclosporin-A (CsA) was given to 22 patients who received allogeneic bone marrow transplants as therapy for aplastic anemia and hematologic malignancies. The drug was given daily for 180 days starting with the day of marrow infusion. Engraftment was not impaired and myelotoxicity was not observed. Cutaneous graft-versus-host disease (GVHD) developed in five patients and all either spontaneously resolved or promptly responded to therapy with steroids. Five patients developed systemic GVHD and all responded to therapy with steroids, but only two survived. Interstitial pneumonia was seen in six patients and was fatal in all of them. Liver function abnormalities were seen in 14 patients but could not positively be correlated with CsA administration. Renal function abnormalities were seen in 17 patients. Amphotericin-B therapy contributed significantly to the renal failure. Serum levels of CsA, measured by radioimmunoassay, could not be correlated with the presence of liver or renal function abnormalities. Overall survival so far has been 50.0%. Second malignancies were not observed, but one patient relapsed with leukemia at 343 days.