ALBERT

Description: Ground level ozone (O3) plays an important role in controlling the oxidation budget in the boundary layer and thus affects the environment and causes severe health disorders. Ozone gas, being one of the well-known greenhouse gases, although present in small quantities, contributes to global warming. In this study, we present a predictive model for the steady-state ozone concentrations during daytime (13:00–17:00) and nighttime (01:00–05:00) at an urban coastal site. The model is based on a modified approach of the null cycle of O3 and NOx and was evaluated against a one-year data-base of O3 and nitrogen oxides (NO and NO2) measured at an urban coastal site in Jeddah, on the west coast of Saudi Arabia. The model for daytime concentrations was found to be linearly dependent on the concentration ratio of NO2 to NO whereas that for the nighttime period was suggested to be inversely proportional to NO2 concentrations. Knowing that reactions involved in tropospheric O3 formation are very complex, this proposed model provides reasonable predictions for the daytime and nighttime concentrations. Since the current description of the model is solely based on the null cycle of O3 and NOx, other precursors could be considered in future development of this model. This study will serve as basis for future studies that might introduce informing strategies to control ground level O3 concentrations, as well as its precursors’ emissions.

Description: Background: Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by isolated low platelet count and a skewed proinflammatory Th1/Th17 profile. However, little is known about the involvement of CD8+ cytotoxic T cells in ITP pathophysiology and whether they are regulated by regulatory T cells (Treg). Immunosuppressive therapy has been the mainstay treatment in ITP. More recently, Thrombopoietin receptor agonists (TPO-RA); Romiplostim (Romi) and Eltrombopag (EPAG), have been increasingly used to stimulate megakaryocytopoiesis to produce more platelets. TPO-RAs are reported to induce complete remission in up to 30% of cases, with limited understanding of their impact on the immune system. Here we describe changes in T cell subsets in patients with ITP: how these changes are affected by disease activity and how TPO-RA may induce remission through modulating the immune system. Methods: Multi-color flow cytometric panels were designed to characterize peripheral blood T cell subsets, including CD8+ T cell and Treg subsets, phenotypically as well as functionally through intracellular cytokine expression. To determine whether CD8+ cells were platelet specific, an IFNγ ELISpot assay was performed using platelets from a healthy donor and PBMC from both HC and patients. Forty patients with ITP were included: 13 were on Romi, 11 on EPAG and 16 on no treatment at the time of analysis. Of these 40 patients, 15 patients had active disease (AD) (platelet-count less than 30 x 109/L) and 25 had stable disease (SD) (〉 30 x 109/L). These patients were compared with 26 age and gender-matched healthy controls (HC). Data were presented as median values; Mann Whitney U and Kruskal Wallis tests were used with Dunn's multiple comparisons correction; a P value of 〈 0.05 was considered significant. Results: CD4/CD8 T cell ratio was significantly lower in patients compared to HC [1.77 vs. 3.97; P value 〈 0.001]. CD45RA+CD62L- Terminally-differentiated CD8+ T cells were significantly higher in patients compared to HC [66.3% vs. 8.56%; P value 〈 0.001]. This finding was more prominent in AD patients than those with SD [66% vs. 44.4%; P value 〈 0.05]. This effector population is polyfunctional, expressing high levels of proinflammatory cytokines including TNFα, IFNγ and Granzyme B when compared to HC [P value 〈 0.05]. Additionally, this population lacks the exhaustion markers PD-1 and Tim-3. Furthermore, these cells were reactive to platelets showing higher IFNγ-producing cells when co-cultured with platelets. CD3+CD4+CD25hiCD127lo Treg frequency did not differ between patients and HC [P value〉0. 05]. Treg functionality was preserved in these patients; no important changes were observed in their capacity to express interlukin2 intracellularly, nor in the cell surface receptor (CD25) [P value 〉 0. 05]. However, Tregs were significantly lower in AD patients with compared to SD patients [2.32% vs. 4.46%; P value 〈 0.05]. Treg function is interactive with other T cell subsets and depends on its relative abundance in relation to other subsets; The Treg/effector CD8+ T cell ratio was significantly lower in patients compared to HC [0.06 vs. 0.16; P value 〈 0.01] and was also significantly lower in AD compared to SD patients [0.03 vs. 0.09 ; P value 〈 0.05]. EPAG-treated patients had a significantly lower effector CD8+ T cells compared to Romi-treated patients [42.4% vs 76.8%; P value 0.05]. Conclusion: While Th1/Th2 cell ratio is often considered as driving ITP, these results demonstrate the involvement of cytokine secreting effector CD8+ T cells in the disease pathogenesis. The imbalance in immune tolerance is also highlighted in the form of a significant reduction in the Treg/effector CD8 T cell ratio. The differences seen in T cell subsets between EPAG- and Romi-treated patients suggest the potential for an additional, differential immunomodulatory effect between the two agents which is currently being explored. Acknowledgment: The authors wish to thank Prof James B Bussel for his insightful comments and support of this work. Disclosures Cooper: Amgen, Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Description: Key Points Human IgM memory B cells possess immunoregulatory properties analogous to transitional B cells. IL-10–producing B cells are deficient in cGVHD.

Description: Abstract 2591 Despite favorable initial responses to induction chemotherapy, most patients with acute myeloid leukemia (AML) will relapse. We hypothesized that just as cancers evade immunosurveillance by suppressing the immune response (“immunoediting”), remission and relapse in AML may be determined by similar immune interactions. After stem cell transplantation natural killer (NK) cells exert powerful allogeneic graft vs. leukemia effects. To explore immunosurveillance by autologous NK cells and immunoediting by AML blasts, we prospectively analyzed NK surface phenotype and function in AML patients at presentation and following remission induction. Using multi-color flow cytometry, we analyzed the surface expression of natural cytotoxicity receptors (NCRs), killer immunoglobulin receptors (KIRs) and C-type lectins directly ex-vivo in 32 consecutive patients at presentation and following complete remission (CR) in 12 patients for whom remission samples were available. Results were compared with 15 healthy controls. NK effector function in AML was measured against K562 leukemia targets and autologous AML blasts by CD107a degranulation and interferon-gamma (IFNγ) and TNF-alpha (TNFα) production. NK cells from 32 patients with AML at diagnosis had an abnormal phenotype compared to controls, with downregulation of the activatory receptor NKp46 (MFI 187± 15 vs. 266± 24, p=0.007) and upregulation of the inhibitory receptor NKG2A (mean 45%± 4.2 vs. 32%± 2.7, p=0.046). Moreover, AML-NK cells were defective in their effector function with significantly reduced CD107a degranulation (5% vs. 11%, p=0.0002), TNFα (1% vs. 3%, p=0.008) and IFNγ production (1% vs. 5%, p= median of 32.6%) at diagnosis were significantly less likely to achieve CR post chemotherapy compared to those with lower NKG2A expression (78% vs. 32% p= 0.041). Furthermore, patients who failed to respond to induction chemotherapy had significantly reduced NK effector function at diagnosis compared to normal controls (mean TNFα production 1% cf. 5% p=0.019). We then sought for evidence of immunoediting by AML on NK cell phenotype and function. Co-incubation of healthy donor NK cells with primary AML blasts for 24 hours resulted in significant reduction in TNFα production (p=0.02), IFNγ production (p=0.01) and a trend to reduced CD107a degranulation (p=0.07) against K562 leukemia targets. Our results indicate that AML blasts can produce long-lasting changes in NK cell subsets and impair their effector function and favouring immune escape from NK cell control. This work justifies larger prospective analyses to relate NK-based prognostic factors to classical predictive factors for remission and relapse, and should guide the development of NK cell based immunotherapy to improve outcome in AML. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2214 Poster Board II-191 Imatinib (IM), nilotinib and dasatinib are remarkably effective as single-agent treatments for chronic myeloid leukemia (CML) in chronic phase (CP). However little is known on their potential impact on the immune system and to date no human in vivo data are available. Data from in vitro and animal studies on the effects of IM on the immune response have been contradictory ranging from impaired antigen-specific T-cell response to enhanced stimulation of tolerant T cells. In addition few data are available to assess potential immunomodulatory effects of the second-generation tyrosine kinase inhibitors (TKIs) nilotinib and dasatinib. Dasatinib has inhibitory activity against a broader range of protein kinases than imatinib including the Src family kinases Lck and Fyn, both of which are associated with T-cell receptor primary signal transduction pathways. Dasatinib may also inhibit B cell signaling through the Lyn pathway which may have potential implications for immunotherapeutic strategies. An understanding of the effects of different TKIs on the immune response will have implications for the development of immunotherapeutic strategies. The aim of this study was to prospectively analyze humoral and cellular immune responses to vaccination against influenza virus (Flu) and Pneumococcus in CP-CML patients treated with IM, dasatinib or nilotinib compared to healthy controls. Fifty CP-CML patients on standard dose TKIs (IM, n=22; dasatinib, n=15; nilotinib, n=13) and 15 healthy controls were vaccinated against Flu (Inflenza vaccine Ph. Eur. 2008/2009, CSL biotherapies) and pneumococcus (Pneumovax II, Sanofi Pasteur MSD). Samples were taken pre and at 1 and 3 months post-vaccination. Titers of IgM and IgG anti-pneumococcal were determined using ELISA technology. A positive response was defined as an IgM serum titer 〉100 U/ml at 1 month; IgG response was considered positive for IgG 〉200 U/ml at 1 or 3 months. To investigate possible correlation between B cell subsets and the pneumococcal humoral response we evaluated IgM memory B cells (CD19+ CD27+ IgMhigh IgDlow) and switched memory B cell (CD19+ CD27+ IgM- IgD-) subsets using flow cytometry. We analyzed the immunological T-cell response to influenza virus both quantitatively and qualitatively using flow cytometry for intracellular TNF-α, IFN-gamma and IL2 and the cytotoxicity marker CD107a. A response was considered positive if there was a minimum of 0.10% Flu-specific TNF-α producing T-cells and the percentage of antigen-specific TNF-α producing T-cells was 2-fold or higher compared to pre-vaccination level. Preliminary results on 28 patients and 11 healthy controls have been analyzed thus far. Significantly fewer patients on TKIs mounted an anti-pneumococcal IgM response (IgM serum titer 〉 100 U/ml) compared to healthy controls (9/28 versus 8/11, p=0.033). An anti-pneumococcal IgM response was detected in 20%, 37.5% and 40% of CML patients on dasatinib, nilotinib and IM respectively, and in 73% of the healthy controls. Moreover, patients on TKI had significantly lower levels of anti-pneumococcal IgM at 1 month compared to healthy controls (median, 84.5 U/ml, range 5 to 200 vs 200 U/ml, range 15 to 200, p=0.006). At 1 month the median levels of IgM in patients on dasatinib, nilotinib and IM were 55 U/ml (range, 12 to 172), 87 U/ml (range, 8-138) and 90 U/ml (range, 5 to 200) respectively. We have so far analyzed CD8 and CD4 T cell responses to Flu vaccination in 15 patients on TKI and 5 healthy controls. Prior to vaccination, T cell responses against Flu were detected in 4/15 patients on TKI and 1/5 healthy controls, indicating pre-existing memory T cell responses to Flu. In these subjects the T-cell response to Flu did not increase significantly after vaccination and as such the response was defined as negative. A significant T-cell response to Flu was seen in 7/15 patients on TKI (median 0.28% TNF-α+CD4+ T cells, range 0.10–2.25%) and in 3/5 healthy control (median 0.79% TNF-α+CD4+T cells, range 0.12–1.34%). These preliminary results suggest that in patients with CML on TKIs the IgM B cell response to vaccination with Pneumovax is significantly impaired compared to healthy controls. We have as yet not detected a significant difference in T-cell response following vaccination with Flu in CML patients on TKIs compared to healthy controls. We are in the process of analyzing the remaining samples. Disclosures: Marin: Novartis: Consultancy, Research Funding.

Description: Abstract 2840 Poster Board II-816 Multiple myeloma (MM) remains incurable with a median survival of 3–4 years. Despite high dose therapy and autologous stem cell transplant (ASCT) most patients relapse with median progression-free survival (PFS) of 2.5–4 years and overall survival (OS) of 4–5 years. Although allogeneic SCT (allo-SCT) is potentially curative due to a graft-versus-myeloma effect, its applicability is significantly limited by high transplant related mortality (TRM). Therefore, the identification of additional independent biological predictors of outcome is required in order to tailor therapy to disease. Natural killer (NK) cells provide first line defence against tumors. NK cells have been shown to recognize and kill myeloma cells both in the allogeneic and autologous settings and donor NK genotype has been shown to influence leukemia free survival following allo-SCT. The aim of this study was to investigate the impact of KIR genotype on event-free (EFS) and OS following ASCT for MM. We performed KIR genotyping on 190 patients with MM receiving a first autologous transplant. KIR genotype and haplotype frequencies were comparable to those published for normal controls. Factors found on univariate analysis to be associated with a shorter EFS included haplotype Bx (containing at least 1 of the KIR B haplotype-defining loci- KIR2DL5, 2DS1, 2DS2, 2DS3, 2DS5, or 3DS1) (median 547 vs 656 days, P = 0.036), ≥3 activating KIR genes (median 547 vs 615 days, P = 0.046), the presence of activating KIR genes KIR2DS1 and KIR3DS1 (median 456 vs 589 days, and 464 vs 619 days, P=0.045 and 0.01 respectively). Disease status at ASCT was the most highly predictive factor for EFS. In patients with good risk disease (CR or PR at ASCT) KIR3DS1 status was highly predictive for EFS 464 days (341–586) vs 731 days (599–862) (P = 0.003) and OS 807days (713-901) vs 967 (925-1009) (P=0.023). KIR3DS1 was not predictive in patients with poor risk disease (P=0.36). Of note KIR3DS1+ve patients were equally represented in good risk (CR and PR) and poor risk (refractory or relapsed) groups at ASCT (around 30% in both groups). Notably the median EFS for KIR3DS1+ good risk patients was not significantly different to poor risk disease patients (P = 0.061). ASCT outcome was then determined according to 3 main groups based on disease status and KIR3DS1 status; A: Good Risk, KIR3DS1-ve; B: Good Risk, KIRDS1+ ve; and C Poor risk (KIR3DS1+ve or -ve). The RR of relapse or death was 1.0, 1.9 (P=0.002, 95% CI 1.3-3.1), and 3.0 (P=0.0001, 95% CI 1.9-4.8) respectively. By multivariate analysis, after adjusting for the presence of adverse cytogenetics and serum albumin and β2m, the KIR3DS1 status and grouping remained highly predictive of poor EFS, RR of 1.0, 2.7 (P= 0.021, 95%CI 1.2-6.2) and 5.3 (P= 〈 0.0001, 95%CI 2.4-11.7) respectively. The prognostic value of KIR3DS1 however, was greatest in patients in whom the ligand for the corresponding inhibitory KIR3DL1, Bw4 was missing. KIR3DS1+ KIR3DL1+ HLA-Bw4 negative patients had significantly reduced median EFS of 400d (315-495) vs 615 (545-684) for all other patients (P=0.048). Again this was most striking in good risk patients. Patients who had the genotype KIR3DS1+ KIR3DL1+ HLA-Bw4 –ve had a significantly shorter EFS survival of 372 days compared to 509 days in KIR3DS1+KIR3DL1+HLA-Bw4+ patients and 793 days for KIR3DS1 negative individuals (P=0.004). In conclusion: Our data from 190 patients with MM suggests that KIR3DS1, a gene previously linked to an increase risk of progression to invasive cervical carcinoma, independently predicts for poor EFS and OS following ASCT. A significant proportion (30%) of patients who are defined as good risk at ASCT (CR and PR) are KIR3DS1+ve and have an EFS which is not significantly different from patients who have refractory/relapsed disease at ASCT. This effect of KIR3DS1 is more significant in the absence of HLA-Bw4, the ligand for the inhibitory receptor KIR3DL1. The mechanism for this is effect is unclear and we are currently performing functional studies to further understand these findings. Disclosures: Apperley: Novartis: Consultancy, Honoraria. Marin:Novartis: Consultancy, Research Funding.

Description: Key Points TKIs impair B-cell immune responses in CML through off-target inhibition of kinases important for B-cell signaling. Our results call for close monitoring of patients on TKI to assess the long-term impact of impaired B-cell function.

Description: CD4+ T cells are important in the establishment of long-lived pathogen-specific immunity. However, the mechanisms by which antigen specific CD4 T resist insult by lymphocytotoxic agents and are sustained long-term is not well defined. A recent report described the existence of a subset of long-lived CD8+ memory T cells with stem-like properties (Turtle et al, 2009), including the ability to efflux cellular toxins through the ABC–superfamily multidrug efflux protein ABCB1. We hypothesized that a similar subset of T cells with drug-effluxing properties also exists within the CD4+ T cell compartment. We used multiparameter flow cytometry to measure the capacity of CD4+ T cells from donors to efflux the fluorescent substrate Rh123. We identified a subset of memory CD4+ T cells with rapid drug-effluxing ability, defined as CD161+CD95+CD45RA-CD127hiCD28+CD25int that shared remarkable phenotypic similarity to CD8+drug-effluxing memory T cells. The stem cell marker c-kit was preferentially expressed on Rh123 effluxing CD4+CD161+ T cells, whereas CD57, a marker of terminal differentiation, was exclusively expressed on non-effluxing CD4+CD161+ T cells. Rh123 effluxing CD4+CD161+ T cells also displayed differential expression of CD31, CD38, CD58, CD122 and IL-18RA. Rh123 effluxing CD4+ CD161+ T cells were undetectable in cord blood, but found in adult blood, consistent with the emergence of this subset of memory T cells as a consequence of antigen exposure during childhood and adult life. We reasoned that this subset may be enriched within the viral-specific T cell repertoire. Indeed, CMV-specific CD4+ T cells were found to share the same phonotypic markers as Rh123 effluxing CD4+CD161+ T cells. We purified CMV-specific CD4+ T cells using the interferon gamma capture assay (Miltenyi), and showed that CMV-enriched CD4+T cells preferentially and rapidly efflux Rh123. The high ABCB1-mediated drug efflux capacity of CD4+ CD161+ memory cells also facilitated their in vitro resistance to daunorubicin, which was abrogated by competitive inhibitors of ABCB1. In keeping with the in vitro data, we found a significant increase in the frequencies of CMV-specific CD4+ T cells in the peripheral blood of patients with AML after recovery from remission induction chemotherapy, suggesting that CMV-specific CD4+ T cells can preferentially survive and proliferate following chemotherapy. Since interleukin (IL)-7 and IL-15 drive the proliferation of T cells during lymphopenia to restore homeostasis, we assessed the response of CD4+CD161+ T cells to stimulation with CD3/CD28 +IL7 and IL15. Both effluxing and non-effluxing sort-purified central and effector memory CD4+CD161+ T cells proliferated and upregulated Ki67 in vitro. Whereas CD4+CD161+ T cells were able to differentiate into CD4+CD161- T cells, a subset retained CD161 expression. These data suggest that although CD4+CD161+ T cells share phenotypic similarities with terminally differentiated cells, they are able to fully proliferate, differentiate to CD161-ve cells and self-renew to preserve the pool of memory T cells CD161 is also a hallmark of Th17 cells. We examined the cytokine profile of CD4+CD161+ T cells stimulated with a pool of overlapping MHC class II CMV pp65 peptides. After 6 and 24 hrs of in vitro stimulation we failed to detect significant IL-17 production. Furthermore, by real time qPCR, the Th1 transcription factor Tbet, rather than RORC2 (a Th17 hallmark), was found to be preferentially expressed in CMV enriched CD4+CD161+ T cells, indicating that CMV-specific CD4+CD161+T cells in fact represent a unique subset of Th1 cells, distinct from Th17 cells. Our data delineate novel findings related to a distinct subset of drug-effluxing CD4+CD161+ viral-specific memory T cells. Signaling pathways leading to CD4+CD161+ABCB1+ differentiation, the role of this subset in drug resistance and the presence or absence of “stemness” which may impart this subset with extended longevity are being explored. †Muharrem Muftuoglu and Abdullah Alsuliman contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.