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Recent observational cohort studies indicated that the incidence of connective tissue diseases such as systemic lupus erythematous (SLE), in adult patients with sickle cell disease (SCD), appears to be increasing. The exact causes underlying this increased risk are still unknown, but patients with SCD are at risk of infections due to immune dysfunction, and a link with regulatory B (Breg) cells is possible as these cells suppress inflammatory responses, maintain tolerance and prevent the development of SLE in animal models via the production of interleukin-10 (IL-10). Herein, we numerically and functionally evaluated Breg cells in a well-defined group of SCD patients. The classification for SCD was based on patients' history, clinical examination, hematological and radiological findings. The American College of Rheumatology (ACR) criteria were used to classify and diagnose SLE patients with or without SCD. Patients were stratified into three groups including SCD patients with SLE (n=21), patients with SCD only (n=24) and patients with SLE only (n=24). Normal healthy individuals (n=24) were used as controls. Circulating levels of Breg cells were prospectively assessed by immuno-phenotyping using specific surface markers, CD19, CD24 and CD38 by flow cytometry. The functional properties were evaluated by IL-10 production and STAT3 phosphorylation after stimulation and cell culture. Unstained cells and n-1 monoclonal antibodies along with live and dead stain were used as controls in all experiments. Comparisons among study groups were performed using ANOVA and unpaired t test, while the Spearman's correlation was used to assess associations. The demographic data were similar among the study groups. All participants were free of infections such as human immunodeficiency virus, hepatitis B and C virus and syphilis. SCD patients with SLE and patients with SLE only were positive for autoimmune markers and showed an average ACR score of five and six respectively. The frequency of Breg cells, as phenotypically defined as CD19+CD24hiCD38hi, ranged between 1.5-8% in healthy controls. The circulating levels of Breg cells were significantly reduced in SCD patients with or without SLE and patients with SLE only when compared to the healthy controls (p Disclosures No relevant conflicts of interest to declare.

Recent data suggests that invariant natural killer T (iNKT) cells, a unique subset of T lymphocytes that express markers characteristic of both T cells and natural killer cells, play a pivotal role in the pathogenesis of sickle cell disease (SCD). Indeed, these cells are being considered as a potential target to prevent or treat acute crises of SCD in adult patients. However, the phenotypic and functional characteristics of circulating iNKT cell subsets in children with SCD remain unknown. Herein, we functionally evaluated iNKT cell subsets in relation to myeloid dendritic (mDC) and plasmacytoid dendritic (pDC) cells and markers of SCD severity in well-defined groups of children with SCD. The classification criteria for SCD was based on the history of patients, clinical examination, hematological and radiological findings. Blood iNKT cell subsets were prospectively studied in 68 children with SCD and normal controls using peripheral blood mononuclear cells, 10-color flow cytometry and culture assays. The iNKT cell subsets were identified by the positive-staining of Vα24Jα18 T cell receptor alpha chain, along with CD3, CD4 and CD8 surface markers and the intra-cellular cytokine production of interferon-gamma (Th1-like), interleukin-4 (Th2-like) and interleukin-17 (Th17-like) cells. The mDC and pDC cells were phenotypically characterized by the expression of HLA-DR, CD123, CD11c, Lin and CD1d, a non-classical molecule that induces the activation of iNKT cells. SCD patients were stratified into three groups including surgically splenectomized (n=23), large spleen (n=21) and steady state (n=24). Comparisons among study groups were performed using ANOVA and unpaired t test, while the Spearman's correlation was used to assess associations. Compared to normal individuals, splenectomized and steady state subjects, large spleen patients exhibited significantly higher levels of CD3iNKT, CD4iNKT and CD8iNKT cells (P=0.04). There were no differences in the levels of iNKT cell subsets between splenectomized and steady state subjects (P=0.56). The levels of mDC and pDC cells were also similar among the study groups (P=0.70). Interestingly, the large spleen patients tend to have higher CD4, but not CD8, Th1-like, Th2-like and Th17-like iNKT cells compared to splenectomized and steady state subjects (p=0.08). The expression levels of CD1d on both mDC and pDC were equivalent among study groups. The levels of CD3iNKT cells were negatively associated with baseline haemoglobin F levels (r=0.45, P=0.04) but not with age, time since splenectomy, spleen size or total hemoglobin levels at phlebotomy. Collectively, these results further advance the functional characterization of circulating iNKT cell subsets in children with SCD and reveal that surgically splenectomized patients have preserved function of iNKT cell subsets that are to be considered for clinical purposes. Disclosures No relevant conflicts of interest to declare.

Abstract 4671 Background: Omega-3 has been reported to enhance the effects of some cancer chemotherapeutic agents and to play a role in the immune system as immunoregulator and immunosuppressant. The effect of a diet rich in omega-3 during immunosuppressed states of anticancer treatment and on bone marrow transplantation (BMT) outcome is not well known. Aims: To study the effect of omega-3 on busulfan-cyclophosphamide (Bu-Cy) conditioning regimen. Moreover, to evaluate the effect of omega-3 on the outcome of BMT after Bu-Cy conditioning. Methods: We evaluated the effects of menhadenfish oil (omega-3 rich) on the myeloablative treatment with Bu-Cy as well as on the outcome of BMT in mice compared to the effects of a diet containing corn oil (omega-6 rich) or to standard chow. Animals were divided into three groups, Group 1, Group 2 and Group 3. In all the groups, female BALB/c mice, 8–12 weeks old, were randomly selected to receive a diet enriched with fish oil, a diet enriched with corn oil or standard chow. The mice were fed for two weeks prior to conditioning. Thereafter, all the mice were injected IP with 80 mg/kg busulfan (Bu) for four days (day –7 to day –4) followed by 200 mg/kg cyclophosphamide (Cy) for two days (days –3 and −2). The mice in groups 1 and 2 were killed on day 0 (the proposed day for transplantation) and spleen and femur bone marrow cells were harvested. Organs were analysed with flow cytometry to evaluate the lymphoid and myeloid cells phenotype. Also, regulatory T cell number and function were studied. Mice in Group 3 were transplanted on day 0 with allogeneic bone marrow cells from 8–12 weeks old male C57BL/6 mice (2.0 × 107 bone marrow cells and 3.0 × 107spleen cells). Results: Fish oil-enriched chow significantly suppressed the function (Figure 1) of CD4+CD25+FoxP3+ regulatory T (Treg) cells compared to standard chow and corn oil fed mice at day 0. However, the percentage of FoxP3+ cells was significantly elevated in mice fed corn oil compared to standard chow. In concert with myeloablative chemotherapy, fish oil enhanced the suppressive effect of Bu-Cy on CD11b+ myeloid and CD11c+CD86+ dendritic cell populations in the bone marrow and spleen compared to the other dietary groups. Recipient mice fed fish oil had lower survival after BMT (Figure 2). A higher survival was observed for corn oil-fed recipients, but this survival rate was still inferior to that of recipients fed standard chow. Feeding recipients omega-3 enriched diet was associated with a more pronounced acute graft versus host disease (aGVHD). BM and spleen from mice fed corn oil demonstrated lower chimerism. Conclusion: Fish oil enhances the immunosuppressive effect of the conditioning regimen (Bu-Cy) in mice. BMT recipients fed a diet rich in polyunsaturated fatty acids either have lower engraftment (corn oil) or react with a lethal aGVHD (fish oil) associated with a suppression of Treg cells. These findings may have clinical implications. PUFA supplementation should be reconsidered and caution is advised in patients undergoing stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

Introduction: Several studies reported the importance of nutrition on SCT outcome. Many food components have been shown to affect the immune system and to interact with a number of drugs used in conditioning and in prophylactic treatment during SCT. Omega-3 fatty acids have immuno-modulatory effects via improving endothelial function, protection against oxidative DNA damage and by suppressing CD4+ T-cell function and proliferation. In the present study we examined the effects of omega-3 on the immune system in mice undergoing SCT. Method: Female BALB/c mice, 8 – 12 weeks, were randomised to receive either diet enriched with menhaden fish oil (omega-3) or, corn oil (rich in omega-6). Both groups were fed for two weeks prior to conditioning (Day 0 was set to be the day of transplantation). Both groups injected intra-peritoneally with 80mg/kg busulfan (Bu) for four days (day –7 to day –4) followed by 200mg/kg cyclophosphamide (Cy) for two days (days –3 & -2). At day 0, half of the mice of both groups were killed and bone marrow, spleen and thymus were harvested and analysed with FACS to evaluate the lymphoid and myeloid cell phenotype. Serum was collected for analysis of cytokines. The remaining mice in both groups were transplanted with allogeneic bone marrow from 8 – 12 weeks male C57BL/6 mice (2.0 × 107 bone marrow cells and 3.0 × 107 spleen cells). Results: Mice fed an omega 3-rich diet showed significant decrease in bone marrow and spleen cellularity compared to that observed in the corn oil group. In particular, the splenic B-cell population was severely affected, showing a 50% decrease in the omega-3 group compared to that seen in corn oil group. The T-cell populations of omega-3 fed mice were also affected, with a significant decrease by 10 to 20 % in the number of CD4+ cells. In contrast, no difference was observed for CD8+ cells, compared to corn oil-fed mice. In the thymus, the number of activated T-cells was decreased by 50% in the omega-3 compared to that observed in corn oil group. NK cells were decreased by 50% in the spleens of omega-3 fed mice. In bone marrow, the myeloid cells (CD11b+) were significantly lower in omega-3 fed mice compared to corn oil-fed controls. All transplanted mice that had received an omega-3 enriched diet died within the first week. These data can be compared to our previous results where more than 60% of mice receiving standard diet survive during the first week and of which at least 20% reach day+21. Conclusion: Omega-3 administration as a dietary supplement for two weeks prior to conditioning regimen showed significant effects on the immune system in terms of a pronounced alteration in the balance of different cells lineages in both bone marrow and spleen. Rapid death of omega-3 fed animals after allogeneic transplantation was observed. Further studies are warranted to investigate the role of omega-3 on the outcome of stem cell transplantation.

Introduction: Polyclonal anti-thymocyte globulin (ATG) is mainly used in hematopoietic stem cell transplantation, as a prevention for graft rejection as well as for GVHD. Immunosuppressive properties of ATG have been considered primarily from the depletion of peripheral lymphocytes. However direct or indirect effects of ATG on other immune components still is controversial. In the present study we evaluated the immunopathologic effects of ATG on immune system in a mouse model. Methods Ten to fourteen weeks old BALB/c and C57BL/6 mice, were injected intra- peritoneally or intravenously with different doses of ATG (0.2mg/kg -25mg/kg) at three dosage schedules. Considering bone marrow transplantation as day 0, ATG were given at days -10, -7 and -5 or -7, -5 and -1 or -5, -3 and -1. At day 0, mice were killed; spleen (SP), bone marrow (BM), lymph nodes (LN) and thymus were removed and analyzed for T-, B-, DC, NK-, T-reg and myeloid cells. To evaluate the efficacy of immunosuppressive effect of ATG, a group of BALB/c mice were conditioned using busulfan (Bu) and ATG and compared to a control group of BALB/c conditioned with Bu (80mg/kg) followed by cyclophosphamide (200mg/kg). Both groups transplanted with BM and spleen cells from C57BL/6 and followed for engraftment and/or GVHD. Results The administration of ATG (0.2- 4.5mg/kg) has resulted in an increase in spleen cellularity while in lymph node and thymus a decreased cellularity was observed. We have found that injecting 4.5mg/kg of ATG at day -7, -5 and -3 significantly decreased T-cell population in spleen and LN compare to Bu-Cy conditioning. The ratio CD4/CD8 increased after ATG treatment showing that CD8 cells are six-fold more sensitive to ATG treatment compared to CD4 lymphocyte. Interestingly T-reg cell population increase after ATG at day 0, however, the increment was negatively correlated with the administration time and positively correlated with the dose. ATG treatment has resulted in an increase in B-cell population by two- and three- fold in spleen and lymph nodes, respectively. Moreover, 1.2- to 3.5-fold increase in DCs, NK and myeloid cells was observed in SP and LN. Thymus cellularity and cell phenotype was less affected while BM cellularity was not affected by ATG treatment. No differences in the ATG effect on cellularity or cell phenotype between IP and IV route was observed. Despite the high immunosuppressive effect observed in T-cell population compared to that seen in Bu-Cy conditioning, no chimerism was observed when Cy was substituted with ATG (4.5 – 10 mg/kg). Conclusion No donor chimerism could be obtained using ATG as a single agent up to 25mg/kg. ATG dose and the administration time are important factors affecting the repopulation of residual T-cells in spleen and lymph node that have to be considered in bone marrow transplantation setting.