ALBERT

Description: Adoptive cell transfer (ACT) of tumor-specific T lymphocytes is a powerful strategy for targeted therapy of cancer, but most of the research in this area has been focused on cytotoxic CD8+ T cells, which directly lyse MHC class I expressing targets. We have recently demonstrated that CD4+ TCR transgenic Th cells specific for self/tumor antigen tyrosinase-related protein 1 (TRP-1) have the ability to reject large established B16 murine melanoma in a model closely mimicking advanced human disease. Moreover, we showed that Th17-polarized cells were more effective in mediating complete tumor rejection than Th1-skewed cells that were capable of producing high quantities of interferon γ (IFN-γ). Interestingly, while Th1 and Th17 populations varied significantly in their phenotype, cytokines profiles, persistence and proliferation patterns in vivo, the Th17 anti-tumor function was critically dependent on the ability of the transferred cells to secrete IFN-γ. This suggests that the Th17 population might gradually acquire Th1-like properties in vivo, and that transcription factors regulating Th17 differentiation (ROR- γt) as well as IFN-γ production and Th1 polarization (t-bet) might be crucial for the effective rejection of the tumor. In order to emulate clinically relevant gene-therapy scenario we inserted TRP-1 TCR into open-repertoire CD4+ T cells from wild-type donors using a retroviral vector. Prior to transduction Th cells were stimulated under neutral (Th0) and polarizing Th1 and Th17 conditions. The majority of transduced cells expressed the Vβ14 chain, released appropriate polarization-defining cytokines upon specific antigenic stimulation in vitro and caused development of massive autoimmune vitiligo upon adoptive cell transfer into wild-type and Rag1−l/&minus mice. Gene-modified cells were readily detectable in Rag1−/− animals by flow-cytometry for more than 4 month after transfer. No off-target GVHD-like toxicities resulting from potential miss-pairing of endogenous and inserted TCR chains were observed. Th0 or Th1 and Th17-polarized TCR-transduced cells were all capable of treating mice bearing large (50–100mm2) B16 tumors, but complete cures with long-term survival occurred more robustly in animals treated with Th17-polarized effectors. To address the question whether plasticity of Th17-skewed effectors is important for their function upon ACT, we treated animals with TCR-transduced Th17-polarized cells derived from t-bet-deficient donors, which are not able to develop Th1-type responses, most importantly, not capable of producing IFN-γ. In contrast to WT-derived Th17 effectors used as a control, t-bet-deficient Th17 cells were able to mediate only minimal delay in tumor growth, suggesting that indeed the ability to acquire Th1-like properties is essential for the anti-tumor function of Th17-skewed lymphocytes. Currently, the clinical effectiveness of the ACT therapy might be hampered by the lack of high-avidity autologous effectors recognizing self/tumor antigens due to the central tolerance mechanisms. Here we demonstrate that the mature effector Th cells can be genetically engineered to express TCR recognizing MHC class II self/tumor antigen and those cells mediate powerful anti-cancer effect in vivo in a realistic model. While tumor-specific Th17-skewed CD4+ T cells are most effective in this setting, t-bet-mediated plasticity in lineage commitment is required for the full therapeutic effect.

Description: Abstract 3283 Donor-derived regulatory T cells (Treg) and natural killer (NK) cells can respectively improve stem cell transplant (SCT) outcome by reducing graft versus host disease (GVHD) severity and exerting a graft-versus-leukemia effect. High frequencies of donor Treg are associated with less GVHD, and low doses of interleukin-2 (IL-2) can expand both NK and Treg after allogeneic SCT. To explore the feasibility of improving the quality of peripheral blood SCT donations, we evaluated the safety and the tolerability of ultra-low dose IL-2 administration to volunteers with the aim of preferentially expanding Treg and NK cells. Twelve healthy volunteers (mean age 34 years; range 22–57) received 0.1 or 0.2 million U/m2/day IL-2 subcutaneously for 5 days (NIH protocol 11-H-0268). Blood samples were collected before and 1, 2, 3, 4, 7 and 28 days after IL-2 injection. Samples were analyzed by multiplex techniques including whole transcriptome gene expression with HumanGene 1.0ST microarrays; serum levels of 69 cytokines and chemokines by Luminex assay; and lymphocyte phenotyping by flow cytometry, to comprehensively characterize the cellular and molecular immune response to IL-2 (“IL-2 immunome”). Treg subsets were determined within the CD4+ T cell population using FoxP3, Helios, CD45RA and CD31 to identify thymus-derived natural Treg (nTreg), induced Tregs (iTreg) and their recent thymic emigrants (RTE). NK cell subsets were determined within CD56+CD3- population using NKG2A, KIR2DL1, KIR2DL2/3, KIR3DL1 and CD57 to identify CD56bright, CD56dim NKG2A+KIR-, and CD56dim KIR+CD57+ cells. All subjects tolerated ultra-low dose IL-2 with minimal adverse events (mainly grade 1–2 injection site reactions). The fraction of FoxP3+Treg in CD4 rose significantly above baseline peaking at 4 days (3.7% vs 5.8%; p=0.0004) after the first dose of IL-2. Treg subset analysis demonstrated that the fraction of nTreg and RTE nTreg in CD4 expanded significantly in the lower dose cohort compared to the higher dose cohort (p=0.004 and p=0.005 respectively). %CD56bright NK significantly increased at 7 days (p=0.008), whereas CD56dimNKG2A+KIR-, and CD56dimKIR+CD57+ NK cells remained at baseline. The Ki67 proliferation marker further verified a significant in vivo expansion of CD56bright NK cells with ultra-low dose IL-2. Cytokine and chemokine profiling demonstrated significant increase circulating level of IP-10 (P=0.0018) through day 2 to 4 after IL-2 injections. In contrast, circulating levels of IL-2, IL-6, IL-10, IL-15 and IL-17 remained unchanged after IL-2 injection. Gene expression microarray studies revealed significant changes in 24 genes (P value 〈 0.1 corrected by false discovery rate (FDR) for multiple testing), including up-regulation of IL-2RA and FOXP3 as early as 2 days after IL-2 injections. Gene Set Analysis (GSA) revealed significant changes (P value 〈 0.1 after FDR) in innate immune response pathways, including Toll-like receptor signaling and interferon signaling. This is the first study to show that ultra-low dose IL-2 could be safely administrated to healthy volunteers to expand thymic-derived natural Treg and CD56bright NK cells. These results raise the possibility of using ultra-low dose IL-2 to boost Treg and NK cells in stem cell donors. Disclosures: No relevant conflicts of interest to declare.

Description: Cluster of differentiation (CD)8+ T cells exist as naive, central memory, and effector memory subsets, and any of these populations can be genetically engineered into tumor-reactive effector cells for adoptive immunotherapy. However, the optimal subset from which to derive effector CD8+ T cells for patient treatments is controversial and understudied. We investigated human CD8+ T cells and found that naive cells were not only the most abundant subset but also the population most capable of in vitro expansion and T-cell receptor transgene expression. Despite increased expansion, naive-derived cells displayed minimal effector differentiation, a quality associated with greater efficacy after cell infusion. Similarly, the markers of terminal differentiation, killer cell lectin-like receptor G1 and CD57, were expressed at lower levels in cells of naive origin. Finally, naive-derived effector cells expressed higher CD27 and retained longer telomeres, characteristics that suggest greater proliferative potential and that have been linked to greater efficacy in clinical trials. Thus, these data suggest that naive cells resist terminal differentiation, or “exhaustion,” maintain high replicative potential, and therefore may be the superior subset for use in adoptive immunotherapy.

Description: CD4+ T helper (Th) cells exist in a variety of epigenetic states that determine their function, phenotype, and capacity for persistence. These polarization states include Th1, Th2, Th17, and Foxp3+ T regulatory cells, as well as the more recently described T follicular helper, Th9, and Th22 cells. Th17 cells express the master transcriptional regulator retinoic acid–related orphan receptor γ thymus and produce canonical interleukin (IL)-17A and IL-17F cytokines. Th17 cells display a great degree of context-dependent plasticity, as they are capable of acquiring functional characteristics of Th1 cells. This late plasticity may contribute to the protection against microbes, plays a role in the development of autoimmunity, and is necessary for antitumor activity of Th17 cells in adoptive cell transfer therapy models. Moreover, plasticity of this subset is associated with higher in vivo survival and self-renewal capacity and less senescence than Th1 polarized cells, which have less plasticity and more phenotypic stability. New findings indicate that subset polarization of CD4+ T cells not only induces characteristic patterns of surface markers and cytokine production but also has a maturational aspect that affects a cell’s ability to survive, respond to secondary stimulation, and form long-term immune memory.

Description: Background T-large granular lymphocytosis (T-LGL) is a rare lymphoproliferative disease characterized by clonal expansion of cytotoxic CD3+CD8+ lymphocytes, presenting with immune mediated cytopenias and associated with autoimmune syndromes. Immunosuppressive therapy (IST) with methotrexate, cyclosporine, or cyclophosphamide can improve the cytopenias in about half the patients but can lead to significant toxicity in older patients. The anti CD52 antibody alemtuzumab is a potent immunosuppressive agent with a good safety profile. We therefore initiated a pilot phase II study to evaluate alemtuzumab as a treatment for T-LGL. Methods 20 consecutive patients with T-LGL were enrolled from October 2006 to August 2012 at National Institutes of Health (www.clinicaltrials.gov - NCT00345345). After a 1 mg test dose, alemtuzumab was administered at 10 mg/dose/day intravenously for 10 days. Peripheral blood, bone marrow, and plasma samples were collected from patients before and at 3 or 6 months after treatment. Blood was analyzed for 1) lymphocytes subsets, T-cell receptor V-beta repertoire and CD57 and CD52 expression by flow cytometry (LSR II, BD, San Jose, California), 2) plasma cytokines using a a magnetic bead based Luminex assay (Affymetrix, CA, USA), 3) STAT3 mutation by direct Sanger sequencing and 4) expression level of 84 genes of the JAK-STAT signaling pathway quantified by PCR array 384 well from SABiosciences (Frederick, MD, USA). Results We report here the results of treatment with alemtuzumab in 20 T-LGL patients enrolled in the first stage of the protocol. Three had associated myelodysplasia (MDS) and two had T-LGL following hematopoietic stem cell transplantation (HSCT). The median age was 61 years (range, 26-82). The median number of prior therapeutic interventions for T-LGL leukemia was 3 (range, 0-7) and the median time from diagnosis to alemtuzumab therapy was 1096 days (range, 18-6054). The median follow-up for all patients is 508 days (range, 99-1481) and for surviving patients 650 days (range, 120-1481). One patient was lost to follow-up 4 months after alemtuzumab therapy. Alemtuzumab was generally well tolerated. Labeled infusion related reactions were common and managed symptomatically. Prolonged and subclinical EBV and CMV reactivations were common but there were no cases of EBV or CMV disease without instituting prophylactic or pre-emptive therapy. Hematological response as defined by protocol was observed in 11 of 20 patients by 3 months after treatment. No patient with MDS or post HSCT responded to alemtuzumab. Four patients relapsed and received a second round of immunosuppression. One patient achieved stable blood counts on cyclosporine, three received alemtuzumab with one patient responding but relapsing 1 year later. STAT3 mutations in the SH2 domain identified in 10 of 20 patients did not correlate with response to alemtuzumab (5 responders and 5 non-responders). Treatment with alemtuzumab reduced significantly the absolute clonal population of T-cytotoxic lymphocytes, as identified by their V-beta receptor phenotype, but they tended to persist in frequency in the peripheral blood of responders. The expanded V-beta clone expressed both CD52 positive and negative cells and both compartments reduced in size after the treatment. When compared with healthy volunteers T-LGL patients had a distinct plasma cytokine signature (IL-12p40, TRAIL, IL22, IP10, MCP1, M-CSF, PDGF-AA, LIF, SCF) as well as JAK-STAT pathway activation prior to treatment but neither was correlated to clinical responses to alemtuzumab, likely due to the various prior IST regimens. Conclusion This is the largest cohort of T-LGL patients treated with alemtuzumab yet reported. Treatment was well tolerated and at this dose minimal side effects were observed. Alemtuzumab treatment in previously heavily treated T-LGL patients results in over 50% response rate and represents a good treatment option for this condition. Disclosures: Off Label Use: Alemtuzumab for T-LGL.

Description: The immune-editing effect of myeloid leukemia has recently been reported in several studies. We previously demonstrated that the K562 leukemia-derived cell line suppresses T cell proliferation, which suggests that myeloid leukemia may function in a similar way to myeloid derived suppressor cells (MDSC). While the mechanism of suppression in leukemia is not fully understood, recent murine and human studies suggest that the STAT3 and arginase pathways play a key role in the immunosuppressive function of MDSC. We hypothesized that myeloid leukemia utilizes the MDSC STAT3 and arginase pathway to evade immune control, and block anti-leukemic immune responses. To evaluate the suppressive capacity of myeloid leukemia on T cell proliferation, we isolated CD34+ blasts and myeloid derived suppressor cells (MDSC: CD11b+CD14+) from blood of primary leukemia samples by FACS sorting (n=5). These cells were co-cultured with CFSE-labeled CD4+ T cells (n=9), previously isolated from healthy donor PBMCs using an automated cell separator (RoboSep). After stimulating with CD3/CD28 Dynabeads (Invitrogen, New York, USA) for 72 hours, proliferation was measured by CFSE dilution of the viable cell population. In three myeloid leukemias studied, CD4+ T cell proliferation was significantly suppressed in the presence of primary CD34 blasts and MDSC cells (p

Description: Abstract 2528 The quantitative lymphocyte recovery after remission induction is known to correlate with the outcome of the patients with acute myeloid leukemia (AML), suggesting the immune-surveillance is at least partly responsible for maintained remissions. However defects in lymphocyte function and increased regulatory T cell numbers suggest a permissive immune environment for leukemia cell growth. Understanding the interplay between the leukemia and the immune system may provide insight into the mechanisms governing leukemia cure or relapse after induction chemotherapy and lead to better remission treatments to prevent relapse. To look for evidence of immune escape and possible immunoediting of lymphocyte function by the leukemia, we undertook an extensive phenotypic characterization of lymphocytes and AML blasts in blood and marrow at presentation, through remission to full hematological recovery. Twenty patients (mean age 57 years; range 31–69) with newly diagnosed AML were enrolled in a protocol permitting blood and marrow collection for investigational purposes (VICCHEM 1073). Mononuclear cells were obtained from blood and marrow samples collected at onset, 14–21 days and 28–35 days after start of induction chemotherapy. Mononuclear cells were analyzed for lymphocyte phenotyping by flow cytometry and subset frequencies were compared with 5 healthy controls (mean age 31). Leukemic blast cells (Lineage CD2/3/19 negative, CD45+ cells) were characterized in blood and marrow. T cells showed several, largely persisting, abnormalities: (1) A decrease in central memory CD197+, CD27+, CD45RO+ CD8+ (p=0.03) and CD4+ T cells (p=0.1) in the blood at presentation with a corresponding decrease in CD197+CD27+ CD45RO-naïve subsets (2) A significant increase in PD1+ high expression suppressor phenotype for CD8+ (p=0.001) and CD4+ (p=0.01) cells at recovery (days 28–35). (3) At presentation FoxP3+ CD4+ regulatory T cells (Treg) were increased significantly at presentation (p=0.03) and remained elevated. This increase was due mainly to augmentation in the HeliosloFoxP3hi induced Treg (p=0.04) and CD45RAloFoxP3hi effector Treg fraction (p=0.002). These changes persisted into remission. The leukemic blast population included a variable population of cells with the CD11b+CD124+HLA-DRlo myeloid derived suppressor (MDSC) phenotype. The frequency of MDSC was higher in bone marrow (median 6.1% vs 3.1% in blood; p=0.44) and a significantly higher proportion of marrow MDSC expressed the lymphocyte suppressor molecule PDL-1 (54.5% vs 28.5%; p=0.0189). NK cell subsets showed significantly lower frequencies of immature CD56bright NK cells (p=0.02), and correspondingly decreased more mature CD56dim CD57-KIR-NKG2A+ subsets at presentation (p=0.039). NK cell subsets trend to recover during remission but with reduced frequency of mature CD57+, KIR+ CD56dim NK cells in recovery (p=0.018). This study confirms the occurrence of significant alterations in T cell phenotype associated with increased regulatory and suppressor phenotype. The occurrence of the MDSC phenotype within the leukemia blast population suggests that myeloid leukemia cells promote a permissive immune environment for leukemia growth and persistence through immunoediting. These changes as well as abnormalities in NK cells tend to persist into remission and may contribute to leukemic relapse. Disclosures: No relevant conflicts of interest to declare.