Increase In Percentage Of CD4 Research Articles

Abstract A008: Intermittent fasting in combination with PD-inhibitor treatment as an innovative treatment strategy in a pre-clinical model of obesity-driven endometrial cancer

Abstract Objective: Endometrial cancer (EC) incidence and mortality continue to rise, with obesity a well-known driver of this disease. Obesity is associated with excess estrogen, hyperinsulinemia, activation of the Akt/mTOR pathway, chronic inflammation and a heightened capacity to harvest dietary energy, which all contribute to the development and progression of EC. Intermittent energy restriction (IER) is thought to have anti-tumorigenic benefits and has been shown to have synergistic effects with chemotherapy and hormonal therapy in various cancer types. IER may also promote anti-tumor immune responses by stimulating CD4+ and CD8+ cells, making this dietary strategy an innovative therapeutic partner with PD-1 inhibitors, which are commonly used in EC. Thus, we evaluated the anti-tumorigenic effects of a switch from high fat diet (HFD) to either IER or low-fat diet (LFD) with PD-1 inhibitor treatment in a mouse model of obesity-driven endometrioid EC. Methods: Lkb1fl/flp53fl/fl mice were fed either HFD or LFD starting at 4 weeks of age. At 12 weeks of age, the HFD-fed mice were randomized to three treatment groups: HFD, HFD-LFD switch or HFD-IER switch. The LFD-fed mice were maintained on LFD. At 16 weeks of age, all mice were injected with AdCre into the left uterine horn to induce EC development. At 26 weeks of age, mice were treated with placebo or a mouse PD-1 inhibitor (BioXCell) (10mg/kg, 2 times/wk, intraperitoneal) for 4 wks (N=8-10 mice per group). RNAseq was performed to determine differences in immune pathways between ECs of obese and lean mice. CD4+ and CD8+ T cells in tumors of obese and lean mice were evaluated by flow cytometry. Results: Obesity resulted in a 52% increase in tumor size, comparing obese versus lean mice (p<0.05). Expression of genes related to T-cell/B-cell receptors, NF-kappa B and TNF pathways were upregulated in the ECs of obese versus lean mice. PD-1 inhibitor therapy increased PD-1 expression on CD4+ and CD8+ T cells in both obese and lean ECs. In addition, PD-1 inhibitor treatment led to an increase in percentage of CD4+ T cells in obese and lean ECs but increased the percentage of CD8+ T cells in only lean ECs. At sacrifice and after PD-1 inhibitor treatment, mean body weights were significantly higher (p<0.05) in the HFD group (42g) than groups receiving LFD (29g), HFD-LFD switch (34g) or HFD-IER switch (23g). EC incidence was lowest in the HFD-IER group (~61%) and highest in the HFD group (~80%); rates were similar in the other diet groups (~75%). In placebo-treated groups, mean tumor weights were significantly lower in mice fed LFD (0.45g), HFD-LFD switch (0.41g) and HFD-IER switch (0.29g) relative to continuous HFD-fed mice (0.94g; p=0.001-0.05). PD-1 inhibitor treatment reduced EC size 24-68% across all diet groups (p=0.05-0.001); size reduction was greatest in the HFD-IER group (0.18g) relative to mice receiving continuous HFD (0.30g), HFD-LFD (0.31g) or LFD (0.31g). Conclusion: IER with PD-1 inhibitor treatment may be an innovative strategy in the management of EC, a highly obesity-driven cancer. Citation Format: Jennifer Haag, Olivia Lara, Chunxiao Zhou, Stephen D. Hursting, Victoria Bae-Jump. Intermittent fasting in combination with PD-inhibitor treatment as an innovative treatment strategy in a pre-clinical model of obesity-driven endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A008.

Abstract P5-17-06: Immune modulation with humanized anti-EGFR antibody panitumumab in an immunocompetent mouse model for inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer and there are no approved targeted therapies specifically for IBC. Targeting the epidermal growth factor receptor (EGFR) pathway is a promising therapeutic target for patients with triple-negative IBC (TN-IBC) with a reported pathological complete response rate of 42% (JAMA Oncology, 2018). The tumor microenvironment (TME) is a critical contributor to the aggressiveness of IBC. Delineating cross-talk between EGFR-targeted therapies and TME components, which define IBC, could inform more efficient combination regimens and novel clinical trial designs for IBC. However, such studies have not been conducted due to the lack of a syngeneic IBC mouse model. Here we report the establishment of an IBC immunocompetent mouse model and the effects of panitumumab (PmAb) on IBC tumor growth and the TME. Methods: TN-IBC cell lines, SUM149 or FC-IBC-02, were mixed with 50% Matrigel and inoculated into mammary fat pads of hu-NSG-SGM3 mice engrafted with hematopoietic stem cells (The Jackson Laboratory). SUM149 tumor growth in hu-NSG-SGM3 mice treated with either IgG2 (isotype control, 4 mg/kg) or PmAb (1 mg/kg and 4 mg/kg) was measured. The percentages of TME components, including human CD4+ T, CD8+ T, regulatory T (Tregs), and natural killer (NK) cells, and M1 or M2 macrophages, in the peripheral blood and tumor tissues treated with IgG2 and PmAb for 7 weeks were measured by flow cytometry. Results: Hu-NSG-SGM3 mice supported the growth of TN-IBC SUM149 and FC-IBC-02 xenografts. These humanized mouse models were named SUM149-huSGM3 and FC-IBC-02-huSGM3, respectively. Analysis of the blood cells showed that SUM149-huSGM3 mice display human CD4+ T, CD8+ T, Tregs, M1 and M2 macrophages. T cell infiltration and M1 and M2 macrophages were also detected in SUM149-huSGM3 tumors. NK cells were not detected in both peripheral blood and tumors. PmAb treated SUM149-huSGM3 mice had significantly reduced SUM149 tumor growth, compared with mice that received the IgG2 control. PmAb treatment increased the percentage of CD8+ T cells and reduced the percentage of Tregs in peripheral blood. A similar analysis of tumor infiltrating lymphocytes isolated from each group showed an increase in percent CD8+ T cells in mice treated with PmAb. There were no significant changes of M1 or M2 macrophages following PmAb treatment. These results suggest that the increase in percentage of CD8+ T cells in peripheral blood and IBC tumors, and the decrease in percentage of Tregs in peripheral blood may contribute to the therapeutic efficacy of PmAb. Conclusion: We established the first immunocompetent mouse model to study the TME and immune response in IBC, which provides the premise for conducting a diversity of novel preclinical therapeutic studies. The mechanism of how immune responses of TN-IBC xenografts mediates the therapeutic efficacy of PmAb in IBC tumors needs to be further investigated. Our study also suggests that combination therapy with immune checkpoint inhibitors may potentiate the efficacy of anti-EGFR therapy in IBC. The therapeutic efficacy of PmAb and anti-PD-L1 combination in SUM149 humanized mice is in progress. Citation Format: Wang X, Shao S, Pearson T, Cheng Y, Reuben JM, Tripathy D, Ueno NT. Immune modulation with humanized anti-EGFR antibody panitumumab in an immunocompetent mouse model for inflammatory breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-17-06.

Abstract 1757: Mobilization of tumor-primed, marrow-infiltrating lymphocytes into peripheral blood with inhibitors of E-selectin or E-selectin and CXCR4

Abstract Marrow-infiltrating lymphocytes (MILs) primed to tumor antigens have been described in patients with hematologic malignancies and in metastatic disease arising from carcinomas. The presence of tumor-reactive MILs in these patients has suggested the possibility of their utilization in T-cell immunotherapeutic approaches. Inherent in this approach are considerations that mediate MIL interactions with the microenvironment and how these may be governed for adoptive or active immunotherapy. Both E-selectin and CXCR4 are known to regulate the homing and retention of T cells to the bone marrow. GMI-1271 and GMI-1359 are potent, small-molecule glycomimetic antagonists of E-selectin and E-selectin/CXCR4, respectively. GMI-1359 is a potent small-molecule glycomimetic dual antagonist targeting E-selectin and CXCR4. In the present studies tumor-specific MILs were established in BALB/c mice that had been induced to reject the syngeneic CT26 colon carcinoma via treatment with anti-CTLA-4 T cell checkpoint antibody, and the subsequent effects of antagonizing E-selectin and/or CXCR4 with GMI-1271 or GMI-1359 on the mobilization and distribution of these bone marrow-derived tumor-specific CD8+ T cells were determined. CT26-immune mice were treated for three days with saline, GMI-1271 (40 mg/kg), or GMI-1359 (40 mg/kg) and 12 hours following the last injection, the phenotype and functional activity of CD8+ T cells were determined in bone marrow and peripheral blood. Additional controls included CT26-immune mice treated with G-CSF (0.125 mg/kg) and tumor-naïve mice treated with saline. Treatment of mice with GMI-1271 and to a greater extent with GMI-1359 led to an approximate 3-4 fold increase in CD8+CD62L+CD44- naïve and CD8+CD62L+CD44+ central memory T cells in peripheral blood. This was not observed following treatment of tumor-immune mice with G-CSF. Treatment of mice with GMI-1271 or GMI-1359 did not affect distribution of CD8+CD62L-CD44+ effector memory T cells in peripheral blood. The increase in percentages of CD8+ naïve and central memory T cells in peripheral blood following treatment with GMI-1271 or GMI-1357 functionally correlated with increased production of IFN-γ ex vivo in response to irradiated CT26 tumor cells or the immunodominant CT26 peptide, AH-1. Collectively these results demonstrate the mobilization or redistribution of marrow-infiltrating tumor-specific CD8+ T cells into peripheral blood as a consequence of E-selectin and/or E-selectin and CXCR4 antagonism. Once in the periphery, these MILs could (1) be collected for adoptive immunotherapy approaches or (2) serve as a systemic augmentation of T cells for combination with immune stimulants as a foundation to boost active immunotherapy. Citation Format: William E. Fogler, Theodore A. Smith, Rachel K. King, John L. Magnani. Mobilization of tumor-primed, marrow-infiltrating lymphocytes into peripheral blood with inhibitors of E-selectin or E-selectin and CXCR4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1757.

Abstract 1688: Enhancement of immune checkpoint PD-1 blockade efficacy in ovarian cancer

Abstract Introduction Advanced stage ovarian cancer is often asymptomatic and only twenty percent of patients with this diagnosis have a survival duration of five years. Immune checkpoint molecule programmed cell death-1 (PD-1) and its ligand, PD-L1, limit T cell immune responses, and thus are immunosuppressive. Clinical trials using anti-PD-1 or anti-PD-L1 antibody treatment has resulted in objective response rates (ORR) in only 10-15% of ovarian cancer patients. Our ultimate goal is to improve this outcome. Recepteur d'origine nantais (RON) is a c-Met related tyrosine kinase which binds to macrophage stimulatory protein (MSP). RON is expressed by myeloid suppressor cells and tumor cells. Inhibition of RON/ MSP ligation upregulates STAT-1 and IL-12 in macrophages, driving IFN-γ production in CD8+T cells and might potentiate the efficacy of anti-PD-1 antibody treatment. Objectives and Methods We hypothesize that combination therapy of anti-PD-1 blocking antibody which restores T cell proliferative and cytotoxic functions, combined with therapy targeting other suppressive pathways, will concomitantly overcome multiple immune suppressive mechanisms, and prevent ovarian cancer in mice. In a pilot study, ovarian cancer was induced in female C57BL/6 (H-2Kb) mice (Jackson Laboratories) at 8 weeks old, by intraperitoneal (I.P.) injection of 1 x 106 ID8-RFP ovarian tumor cells in 5 mice/ group. We determined the efficacy of anti-PD-1 antibody (RMP1-14, BioXCell; 200ug/ dose, 0.5 ml vol. I.P., 4 doses) given alone or with a RON inhibitor, BMS-777607 (each dose 50 mg/ kg body weight, 20 doses orally over 5 weeks; Selleck Chemicals). Mice were euthanized at about day 70 when control mice with disease had extended abdomens. Results Treatment of mice with an anti-PD-1 blocking antibody combined with a RON inhibitor resulted in disease improvement in 5/5 mice. The average volume of ascites recovered from 5 combination treated mice was 1.62 ml (2/5 mice had no ascites) compared with 6.64 ml from the corresponding control (a 4-fold reduction in ascites with combination treatment). There was an average 2 fold reduction in ascites volume in anti-PD-1 antibody treated mice in comparison with IgG controls (4.14 ml versus 8.62 ml). BMS-777607 treatment (vs vehicle) did not significantly alter ascites volume. Flow cytometry evaluation of spleen cells showed that in the combination treated mouse group, there was an increase in the average percentage of CD3 (combined treatment group 26.7 vs control 8.5%), CD4 (10.6 vs control 5.0%) and CD8 T cells (10.6 vs control 2.7%). We are investigating changes in immune responses genes in RNA of intestines and livers. Conclusions Anti-PD-1 antibody treatment is effective in treating ovarian cancer in mice, but treatment with this agent combined with RON/ c-Met inhibitor, BMS-777607 is superior to either single therapy. Combination treatment with these 2 agents holds promise as a novel therapeutic approach for ovarian cancer. Citation Format: Maureen Drakes, Swati Mehrotra, Ronald Potkul, Yueying Liu, M. Sharon Stack, Patrick Stiff. Enhancement of immune checkpoint PD-1 blockade efficacy in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1688.

6-Mercaptopurine modifies cerebrospinal fluid T cell abnormalities in paediatric opsoclonus-myoclonus as steroid sparer.

The purpose of this study was to evaluate the capacity of 6-mercaptopurine (6-MP), a known immunosuppressant, to normalize cerebrospinal fluid (CSF) lymphocyte frequencies in opsoclonus-myoclonus syndrome (OMS) and function as a steroid sparer. CSF and blood lymphocytes were immunophenotyped in 11 children with OMS (without CSF B cell expansion) using a comprehensive panel of cell surface adhesion, activation and maturation markers by flow cytometry, and referenced to 18 paediatric controls. Drug metabolites, lymphocyte counts and liver function tests were used clinically to monitoring therapeutic range and toxicity. In CSF, adjunctive oral 6-MP was associated with a 21% increase in the low percentage of CD4+ T cells in OMS, restoring the CD4/CD8 ratio. The percentage of CD4+ T cells that were interferon (IFN)-γ+ was reduced by 66%, shifting the cytokine balance away from T helper type 1 (Th1) (proinflammatory) predominance. The percentage of natural killer (NK) cells decreased significantly in CSF (-32%) and blood (-67 to -82%). Low blood absolute lymphocyte count was more predictive of improvement in CSF lymphocyte proportions (correlated with % CD4+ T cells) than the 6-thioguanine level (no correlation). 6-MP was difficult to titrate: 50% achieved the target absolute lymphocyte count (<1·5 K/mm); 20%, the 'therapeutic' 6-thioguanine level; and 40% the non-toxic 6-methylmercaptopurine level. Side effects and transaminase elevation were mild and reversible. Clinical steroid-sparing properties and lowered relapse frequency were demonstrated. 6-MP displayed unique pharmacodynamic properties that may be useful in OMS and other autoimmune disorders. Its steroid sparer capacity is limited to children in whom the therapeutic window can be reached without limiting pharmacokinetic factors or side effects.

Influence of Immunogenicity of Allogeneic Bone Marrow Mesenchymal Stem Cells on Bone Tissue Engineering.

Allogeneic bone marrow mesenchymal stem cell (allo-BMSC)-based tissue-engineered bone (TEB) has great potential for bone defect repair. However, the immunogenicities and biological roles of allo-BMSCs are still controversial. In this study, we established an animal model of critical-sized mandibular defect in beagle dogs and compared the repairing effects of allo-BMSC-based TEB with autogenic BMSC (auto-BMSC)-based TEB without the administration of immunosuppressants. During the first 2 weeks postimplantation, a transient immune response in the allo-BMSC group was detected with an increase in proinflammation cytokines TNF-α, IFN-γ, and IL-2, a declination of anti-inflammation cytokine IL-10, and an increase in percentages of CD4(+) and CD8(+) T-cell subsets in peripheral blood. Nevertheless, there was no significant difference in bone union achievement, bone mineral density, and biomechanical properties between the two groups at 12 and 24 weeks postimplantation. Further subcutaneous implantation of allo-BMSCs/scaffold also exhibited the similar transient immune responses in the first 2 weeks postimplantation but followed by a decreased bone formation at 4 and 8 weeks postimplantation. These findings indicate that allo-BMSCs can induce a transient immunoreaction, which may temporally delay the osteogenesis of allo-BMSC/scaffold complex in early stage of in vivo implantation, whereas the long-term engineered bone formation was not affected.

Expressions of interleukin-9 and the transcription factor PU.1 in peripheral blood of patients with chronic urticaria and their significance

Objective To investigate the relationship of interleukin-9（IL-9）and the transcription factor PU.1 with chronic urticaria. Methods Thirty patients with chronic urticaria（patient group）and 30 healthy volunteers（control group）were included in this study. Venous blood samples were collected from these subjects. Enzyme-linked immunosorbent assay（ELISA）was performed to detect serum levels of IgE, flow cytometry（FCM）to determine the percentage of CD4+IL-9+ T cells, real-time fluorescence-based quantitative PCR（RTFQ-PCR）to measure the mRNA expressions of IL-9 and PU.1 in peripheral blood mononuclear cells（PBMCs）. Measurement data were compared between the two groups by independent-samples t test, and relationship among these parameters was assessed by Pearson correlation analysis. Results The expressions of IL-9 and PU.1 mRNAs in patients with chronic urticaria were significantly higher than those in healthy controls（4.44 ± 1.90 vs. 3.20 ± 1.78, t = 2.60, P < 0.05; 3.26 ± 1.59 vs. 2.34 ± 1.47, t = 2.34, P < 0.05）. Compared with the control group, the patient group showed increased percentage of CD4+IL-9+ T cells in peripheral blood（0.63% ± 0.44% vs. 0.22% ± 0.12%, t = 5.04, P < 0.01）and serum IgE levels（（82.04 ± 31.72）vs.（60.74 ± 28.26）IU/ml, t = 2.75, P < 0.01）. The percentage of CD4+IL-9+ T cells and levels of IL-9 and PU.1 mRNAs in peripheral blood were all positively correlated with serum levels of IgE in these patients（r = 0.596, 0.767, 0.746, respectively, all P < 0.01）. Conclusion T helper type 9（Th9）cells and IL-9 may take part in the occurrence of chronic urticaria. Key words: Urticaria; Interleukin-9; Th9 cells

The influence of phytoncides on the immune system of broiler chickens and turkeys.

The aim of our study was to investigate the influence of adiSalmoSOL PF dietary supplement, given for 3 days in drinking water, on selected parameters of cell-mediated (Experiment I) and humoral (Experiment II) immunity in chicken and turkey broilers. In Experiment I, birds were randomly divided into two groups of 10 birds each. Group 1 comprised control turkeys or chickens, whereas group 2 birds were administered adiSalmoSOLPF. In Experiment II, a total of 69 chickens were divided into three groups (1-3) of 23 birds each. At the age of 25 days, group 1 was given adiSalmoSOLPF. Birds from groups 1-3 were vaccinated at 28 days of age. Group 2 was given adiSalmoSOLPF after vaccination. In Experiment I, a significant increase in percentages of CD4 + T lymphocytes in the bursa of Fabricius, cecal tonsils and spleen, CD8 + T lymphocytes in the thymus and spleen, CD4 + CD8 + T lymphocytes in the bursa of Fabricius and ileal mucosa and IgM + B lymphocyte in the ileal mucosa were observed in group 2 chickens. In Experiment I, a significant increase in percentages of CD4 + T lymphocytes in the thymus and spleen, CD8+ T lymphocytes in the cecal tonsils and blood, and CD4 + CD8 + T lymphocytes in the thymus and ileal mucosa was recorded in group 2 turkeys. No differences in percentages of IgM + B lymphocytes were observed between turkey groups. In Experiment II, the highest post-vaccination titers of anti-IB antibodies were observed in group 2, but it was not statistically significant. The results of our study indicate that adiSalmoSOLPF showed immunomodulatory activity in chickens and turkeys.

CD5-CK2 binding domain regulates TCR signals and apoptosis during thymic development. (HEM4P.232)

Abstract CD5 has been previously shown to play an important role in thymic development by downregulating TCR signaling. The receptor contains two major signaling domains in its cytoplasmic tail. The first domain is a functional ITIM domain that recruits proteins such as Ras-Gap, PI3K, Zap 70 and others. The second domain is a CKII Binding Domain (CD5-CKII BD), which in peripheral T cells promotes survival, proliferation and Th differentiation. However, the role of these CD5 signaling domains in early T cell development is poorly understood. In this work we determined the function of CD5-CKII BD in differentiation of thymocyte subpopulations, activation of TCR downstream molecules and regulation of apoptosis using a knock-in mouse in which the CK2 BD is selectively ablated (CD5ΔCKII-BD). Our data show that in non TCR-Tg and in OTII TCR-Tg the loss of CD5-CKII signaling results in a decrease in DP populations in proportion and total cell number and an increase in percentage of CD4+ thymocytes. This reduction in DP thymocytes of OTII.CD5ΔCKII-BD mice was associated with increase in antigen-induced acute activation of ERK phosphorylation relative to OTII.wild-type mice. Additionally, DP and CD4+ thymocytes in CD5ΔCKII-BD mice had increased apoptosis along with elevated levels Caspase 3. Collectively, these results demonstrate that CD5-CKII BD is an important regulator of TCR signals during thymic development and also is a major regulator of the apoptotic events in the thymus.

Evaluation Of CD33 Expression and Functional Analysis Of The CD33/CD3 Bispecific BiTE® Antibody AMG 330 In Primary AML Samples

Antibody-based immunotherapy represents a promising strategy to specifically target and eliminate chemoresistant leukemic cells in acute myeloid leukemia (AML). We evaluated a single-chain bispecific CD33/CD3 BiTE® antibody (AMG 330) for its suitability as immunotherapy in AML. A prerequisite for successful immunotherapeutic approaches using this molecule is the expression of CD33 on AML blasts including leukemic stem cells (LSCs). Therefore, we quantified CD33 expression on AML blasts and LSCs by flow cytometry (mean fluorescence intensity, MFI) and correlated expression intensity with cytogenetic and molecular disease characteristics in order to identify patient cohorts possibly most suited for CD33-targeted therapies. CD33 expression was detected in >99% of patient samples (n=621, MFI ratio ≥ 1.5) although highly variable. A strong correlation between high CD33 expression levels and NPM1 mutations (p<0.001) was found. In contrast, low CD33 expression levels were significantly associated with complex karyotypes and t(8,21) translocations (p<0.001). Furthermore, LSCs within the CD34+/CD38- compartment displayed CD33 at higher levels than healthy donor stem cells (p=0.047). Importantly, colony formation of CD34+/Lin-cells from healthy donors was not affected after pre-incubation with AMG 330 and T-cells.A major hurdle for measuring cytotoxic effects on AML blasts has long been that primary AML patient samples show progressive cell death within a few days ex-vivo. To simulate the natural setting of target and T-cells in AML patients, we developed a long-term culture system for AML blasts that allowed us to observe these co-cultures for up to 5 weeks. Thus, we were able to show effective elimination of AML blasts within primary samples by AMG 330-activated and expanded residual CD3+/CD45RA-/CCR7+ memory T-lymphocytes. While the functional relevance of CD33 expression levels was shown by faster lysis kinetics of CD33BRIGHT vs. CD33DIM AML cell lines in an in-vitro cytotoxicity assay potent anti-leukemic activity on primary AML blasts was observed irrespective of CD33 expression level. At low effector to target ratios (up to 1:79), the recruited T-cells lysed autologous blasts completely in the majority of samples. Further T-cell analysis showed that naive T-cells (CD45RA+/CCR7+) were not expanded by AMG 330; neither were terminally differentiated T-cells (CD45RA+/CCR7-), probably due to their poor proliferative capacity. We did not observe an increase in percentage of CD3+/CD4+/CD25+/FoxP3+regulatory T-cells in the presence of AMG 330, suggesting that these cells may not have impacted AMG 330-mediated T-cell activity in our experiments. Compared to control cultures, T-cells were shown to up-regulate the activation markers CD25, PD-1, TIM3 and LAG3 upon response to AMG 330, which was partially reversible after complete target cell elimination. However, PD-1 up-regulation did not correlate with an up-regulation of PD-L1 on AML blasts despite substantial INFγ secretion by activated T-cells.This study provides the first correlation of CD33 expression levels to a comprehensive genotype analysis in adult AML patients. While CD33 expression may vary by AML biologic subgroup, AMG 330 exposure led to lysis of AML blasts even in samples with low levels of expression. Targeting CD33 using AMG 330 in primary AML samples led to efficient T-cell activation and expansion as expected from the mechanism of action of BiTE® antibodies. The remarkable ex-vivo activity of AMG 330 supports further development of AMG 330 as an immunotherapy for patients with AML. Disclosures:Kufer:AMGEN Research (Munich) GmbH: Employment; AMGEN Inc.: Equity Ownership. Kischel:AMGEN Research (Munich) GmbH: Employment; AMGEN Inc.: Equity Ownership. Zugmaier:Amgen Research (Munich) GmbH: Employment; Amgen Inc.: Equity Ownership. Baeuerle:AMGEN Research (Munich) GmbH: Employment; AMGEN Inc.: Equity Ownership. Riethmüller:AMGEN Inc.: Equity Ownership.

An Expansion of CD3+CD8+CD28-NKG2A+ T-Cells Occurs in Individuals With Active Celiac Disease

Background: Celiac disease (CD), characterized by small intestinal enteropathy, occurs in genetically susceptible individuals intolerant to gluten. Activation of the innate and adaptive immune system occurs locally in the small bowel mucosa. However, recent studies have also described the presence of Gliadin specific T-cells in the peripheral blood of individuals with CD. An oligoclonal expansion of CD8+ T-cells bearing the inhibitory NK-cell receptor NKG2A has been shown to be a marker of antigen experienced cytolytic T-cells in small bowel mucosa and the peripheral blood. Thus, In this study, we characterized the phenotype of circulating T-cells in CD patients during different disease phases, in comparison to normal individuals, to discern disease specific alterations. Methods: Peripheral blood samples were taken at the time of endoscopy from patients in each disease phase (active celiac disease ACD, gluten free diet GFD), as well as normal individuals. The frequency ofCD4+and CD8+ T-cells expressing the NK-cell activating (NKG2D) and inhibitory (NKG2A) receptors was evaluated in conjunction with CD28 expression by flow cytometry of peripheral blood mononuclear cells (PMBC). Results: A total of 15 peripheral blood samples from age and gender matched individuals with ACD (n=5), those on GFD (n=5), and normal controls (n=5) were analyzed. The mean age of the subjects was 35.4yrs and 80% were female. All phases of CD were associated with an increased percentage ofCD8+CD28T-cells compared to age matched controls (P=0.038 -for ACD compared to controls, and 0.082 for GFD compared to controls). Further characterization for NKG2A and NKG2D expression revealed an expansion of NKG2A+ CD28T-cells in ACD compared to controls (p=0.014). The percentage of this subset decreased to control levels in individuals on GFD (p=0.427). No significant differences in CD4+NKG2A+CD28or NKG2D+CD28T-cell subsets was observed between CD patients and controls. CD8 as well as CD4 T-cells from patients with ACD, but not those on GFD, exhibited spontaneous proliferation In Vitro. This proliferating response can further amplified by addition of IL-15 and/or Gliadin. Conclusion: Our findings suggest changes in subsets of circulating CD8+ T-cells in ACD patients that might be of functional importance. It is presently unclear whether the expansion of CD8+CD28-NKG2A + T-cells represents a Gliadin-specific population with effector cytotoxic or regulatory potential. It is conceivable that NKG2A up-regulation on T-cells represents a homeostatic response to dampen the immune response to offending antigens. Further studies evaluating the antigen specificity and cytokine profiles of this subset will shed more light on the systemic immune response in individuals with untreated celiac disease.

Effects of immunostimulatory CpG ODN on experimental allergic conjunctivitis caused by aspergillus fumigatus

Background Researches demonstrated that CpG ODN,a immunostimulatory sequences,has preventing and treating effect on allergic conjunctivitis caused by protein allergen.However,its effect on allergic conjunctivitis caused by fungal allergen is unclear. Objective This study aimed to investigate into whether the Th1-Th2 switching immunostimulatory CpG ODN could reverse the response in the murine allergic conjunctivitis model caused by aspergillus fumigatus. Methods A mixture of spores and hyphae of aspergillus fumigatus strain was used to induce allergic conjunctivitis in male BALB/C mice aged 6-8 weeks.This experiment was designed into preventive or therapeutical treatment program.Under both settings,allergic conjunctivitis of the animals were treated with CpG ODN,nonstimulatory GpC ODN or PBS.After the last challenge with the allergen,the clinical symptoms of the animals were scored based on the criteria of Magone.The animals were sacrificed and the histopathological examination of conjunctiva was performed.Expression of TLR4 mRNA in conjunctiva was analyzed by real-time PCR assay.The responsiveness and populations of lymphocytes in spleen and draining lymph nodes were analyzed by flow cytometry.The use complied with the Standard of Association for Research in Vision and Ophthalmology. Results In the prevention mode.CpG ODN decreased subconjunctival infiltration compared with GpC ODN and PBS groups with the average neutrophil count index(21.25 ±11.59/section,30.75 ±11.44 section and 69.00±9.90/section,respectively).Expression of TLR4 mRNA was up-regulated significantly by CpG ODN.The clinical scores for CpG ODN group were insignificantly lower than those in GpC ODN group and PBS group(P＞0.05).In the therapeutic mode,compared with GpC ODN and PBS groups,the allergic symptom score in CpG ODN group manifested significantly lower(t=4.000.t=2.750,P＜0.01)and showed fewer cellular infiltration(t=4.870,t=3.829,P＜0.01)and higher expression of TLR4 mRNA(P＜0.01).In cultured splenic and draining lymph node cells,increased percentages of CD4+ CD25+ and CD4+ CD25+ CD69+ in CpG ODN group were observed compared with control groups(|P＜0.05). Conclusion CpG ODN can relieve aspergillus fumigatus-induced allergic conjunctivitis via either subconjunctival injection or topical application by upregulating expression of TLR4 and activating Treg lymphocytes. Key words: Aspergillus fumigatus; Allergic conjunctivitis; CpG ODN; T-Lymphoeytes; Regulatory

2-color analysis of lymphocyte subpopulation of bronchoalveolar lavage fluid and peripheral blood in patients with sarcoidosis

By means of 2-color analysis, we investigated the lymphocyte subpopulation of bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) of 13 patients with pulmonary sarcoidosis. The BALF-CD4+ T cells of normal subjects and patients with sarcoidosis consisted almost completely of CD4+4B4+ cells (helper inducer T cells) and BALF-CD8+ T cells were almost completely composed of CD8+CD11- cells (cytotoxic T cells). The BALF findings of sarcoidosis were characterized by increased percentages of CD4+4B4+ cells (54.9 +/- 15.7%), decreased percentage of CD8+CD11- cells (12.0 +/- 8.4%) and increased percentage of CD4+HLA-DR+ cells (27.8 +/- 13.1%). These findings suggest that activated CD4+4B4+ cells (helper inducer T cells) may primarily contribute to the pathogenesis of pulmonary sarcoidosis. The in vivo or in vitro functions of CD4+4B4+ cells in sarcoidosis need further investigation.

T-lymphocyte subsets and eosinophil counts in acute and convalescence chickenpox infection: a household study in Guinea-Bissau

Objective. To investigate changes in T-lymphocyte subsets, CD4+ and CD8+ lymphocytes, WBC, lymphocytes and eosinophil granulocytes during the acute and the convalescence phase of chickenpox infection. Methods. During an epidemic of chickenpox, a household study was performed in a semi-urban area of Bissau, Guinea-Bissau. Varicella antibodies were determined to assess diagnostic certainty. To determine the timing of changes, haematological markers and T-cell subsets (immunocytochemical method) were analysed in the acute phase, 0–9 days after the rash, and in the convalescence phase, 35–45 days after the rash. Results. In the acute phase, the CD4 percentage, CD4/CD8 ratio, and neutrophil percentage declined, whereas the CD8 percentage, WBC, CD4 and CD8 counts, and the lymphocyte percentage increased over the same period, most markedly for the CD8 count. The eosinophil percentage increased significantly with time from onset of rash. Between acute and convalescence samples there was an increase in CD4 percentage, CD4/CD8 ratio, and CD4 count, and a marked decrease in CD8 percentage and CD8 count. The changes were not significant for WBC, lymphocyte percentage, neutrophil percentage, and monocyte percentage, but eosinophil percentage was significantly increased 5–7 weeks after the onset of rash. The haematological changes were related to number of pox and intensity of exposure; a high eosinophil percentage was associated with less severe disease, i.e. less pox. Conclusion. We report significant changes in T-lymphocyte subsets during the acute phase of chickenpox infection, including a suppression of CD4+ T-cells and an augmentation of CD8+ T-cells. The levels were normalized 1 month later except for eosinophils, and we found no persistent CD4 suppression after chickenpox. An increased number of eosinophils in the peripheral blood was demonstrated early in the acute phase of the disease, and remained elevated in the convalescence phase.

NOD/LtSz-Rag1nullPfpnull mice: a new model system with increased levels of human peripheral leukocyte and hematopoietic stem-cell engraftment.

A critical need exists for effective small-animal models that accept engraftment of human hematopoietic progenitor cells and mature lymphocytes. The purpose of this study was to determine the phenotypic effects of perforin (Pfp) deficiency on nonobese diabetic (NOD)-Rag1null mice and to evaluate the ability of NOD/LtSz-Rag1nullPfpnull recipients to support engraftment with human hematolymphoid cells. A new genetic stock of NOD mice doubly homozygous for targeted mutations at the recombination activating gene (Rag)-1 and Pfp genes was developed. NOD/LtSz-Rag1nullPfpnull mice were studied for immunopathologic and hematologic abnormalities. The ability of these mice to support engraftment with human peripheral blood mononuclear cells (PBMC) and umbilical-cord blood hematopoietic progenitor cells was assessed. NOD/LtSz-Rag1nullPfpnull mice lacked mature B cells, T cells, natural killer (NK) cell cytotoxic activity and were devoid of serum immunoglobulin (Ig) throughout a 37-week lifespan. These mice supported heightened engraftment with human PBMC as compared with NOD/LtSz-Rag1null controls as evidenced by a 4- to 5-fold increase in percentages of human lymphocytes and a 7- to 13-fold increase in percentages of CD4+ T cells in the peripheral blood and spleen. Total numbers of human CD4+ T cells were increased approximately 20-fold in the spleens of NOD/LtSz-Rag1nullPfpnull mice. These mice also showed approximately 12-fold higher levels of engraftment with human umbilical-cord blood cells compared with NOD/LtSz-Rag1null mice. NOD/LtSz-Rag1nullPfpnull mice are devoid of mature B cell, T cell, and NK cell cytotoxic activity, engraft at high levels with human PBMC, and hematopoietic progenitor cells and provide a new NK cell-deficient model for human hematolymphoid cell engraftment.

Characterizing immune reconstitution after long-term highly active antiretroviral therapy in pediatric AIDS.

In this study, we sought to characterize the T lymphocyte recovery in vertically HIV-1-infected children who respond to long-term highly active antiretroviral therapy (HAART). A 3-year longitudinal retrospective study was used to perform a cross-sectional study of 32 children rated according to the time course of CD4(+) T cell percentages in response to antiretroviral therapy and CDC clinical classification: (1) long-term asymptomatic (LTA group): 8 children in A1 during the whole follow-up period; (2) responsive to HAART (Rec group): 13 children in C3 before HAART who achieved CD4(+) T cell counts of > 500 cells/mm(3) after 3 years of HAART; and (3) nonresponsive to HAART (Non-Rec group): 11 children in C3 during the whole follow-up period despite 3 years of HAART. We also studied 17 healthy age-matched uninfected children as controls. Lymphoproliferative responses (LPRs) were evaluated by incorporation of [(3)H]thymidine, identification of T cell subsets by three-color flow cytometry, and determination of thymic production of T cells by quantification of T cell receptor rearrangement excision circles (TRECs). Interestingly, the Rec group showed an increase in percentage of CD4(+) T cells and a decrease in viral load, and recovered LPRs to mitogens and recall antigens, with values similar to those of the LTA group. Moreover, the Rec group produced similar percentages and absolute counts of naive (CD45RA(+)CD62L(+)) CD4(+) and CD8(+) T cells, and TRECs similar to those of the LTA group. In particular, the Rec group produced similar percentages of CD8(+)CD28(-)CD57(+) and CD8(+)CD28(-)CD57(-) T cell subsets compared with controls. Our data indicate that among children who have already progressed to AIDS and severe immunodeficiency but who respond to HAART, the immune system can recover and resemble those of nonprogressors or even uninfected children, in quantitative as well as in functional terms.

Effects of dietary conjugated linoleic acid in nursery pigs of dirty and clean environments on growth, empty body composition, and immune competence.

Early-weaned pigs (n = 64) averaging 5.3 +/- 0.3 kg and distributed into two environments (dirty and clean) were used to evaluate effects of conjugated linoleic acid (CLA) on growth performance, immune competence, and empty body composition. A factorial (2 x 4) arrangement within a split-plot design, with four littermate pigs as the experimental unit for the environment, pig within litter as the experimental unit for dietary treatment, and d-0 body weight used as covariate, were used in data analysis. Diets were formulated to contain CLA at 0, 0.67, 1.33, or 2% and to exceed the NRC (1988) nutrient needs of pigs. Animals were given ad libitum access to feed for 7 wk in three phases (I, 1 to 2; II, 3 to 5; and III, 6 to 7 wk). Within phases, diets were isocaloric and isonitrogenous. In Phase I, as dietary CLA concentration increased, ADG and ADFI decreased linearly (P < 0.05 and P < 0.02, respectively). In Phase II, upon adaptation to dietary CLA supplementation, ADG increased quadratically (603, 623, 622, and 548 g/d; P < 0.01), ADFI decreased linearly (873, 840, 867, and 717 g/d; P < 0.02) and gain:feed ratio tended to increase linearly (691, 742, 715, and 763; P < 0.07). In Phase III, no differences in growth performance were attributed to either dietary or environmental treatments. The poor health status associated with the dirty environment induced a growth suppression; pigs in the clean room had a greater cumulative ADG (P < 0.01) and ADFI (P < 0.01) than pigs in the dirty room. In Phase I, lower plasma urea nitrogen levels observed in pigs found in the dirty room (P < 0.03) indicated a lower protein intake caused by a lower ADFI. The effects of dietary CLA on peripheral phenotypic profiles of lymphoytes did not appear until d 42. However, as indicated by the growth suppression of pigs in the dirty room, the negative effects of the environmental challenge on pig health and growth had already appeared during phase I. On d 42, CLA induced a linear increase in percentages of CD8+ lymphocytes (21.7, 22.3, 28.0, and 32.7%; P < 0.001). These data suggest that a 42-d dietary CLA supplementation preceding a disease challenge could have prevented disease-associated growth suppression. Also, CLA-mediated amelioration of particular infectious diseases will depend on which CD8+ T cell subset (i.e., CD8alphaalpha-immunoregulatory or CD8alphabeta-cytotoxic) is most influenced by dietary CLA supplementation.

Low-Dose Daily Subcutaneous Interleukin-2 in Combination with Highly Active Antiretroviral Therapy in HIV + Patients: A Randomized Controlled Trial

Purpose: Previous studies with intermittent interleukin-2 (IL-2) therapy using intermediate and high levels of IL-2 have demonstrated significant increases in the CD4 + T cell count in HIV-infected patients. Intermittent regimens are amenable to outpatient use, but severe adverse events are frequently experienced with intermediate- and high-dose levels of IL-2. Therefore in this study, the effect of daily, subcutaneous low-dose IL-2 therapy on safety and immunological endpoints was investigated to determine whether immunological benefit could be achieved without toxicity in HIV-infected patients also receiving highly active antiretroviral therapy (HAART). Method: A total of 115 patients were enrolled in the trial. Fifty-six asymptomatic HIV-infected patients who had CD4 + T cell counts less than 300 cells/μL at screening and a stable HIV viral load received low-dose IL-2 (1.2 million IU [MIU]/m 2 beginning dose) once daily in conjunction with HAART (IL-2 group). Fifty-nine patients received HAART alone (control group). Results: A dramatic effect of IL-2 on the natural killer (NK) cell population was observed with mean increases of 156 cells/μL in the IL-2 group compared to 19.93 cells/μL in the control group (p < .001). Additionally, IL-2-treated patients experienced a statistically significant increase in the mean percentage of CD4 + T cells (3.52% increase) when compared to control patients (1.33% increase) (p < .001). The expanded CD4 + T cell population was primarily of the naive phenotype, with mean increases of 4.53% for the IL-2 group and 0.31% for the control group (p < .001 for between-group difference). In addition, a higher proportion of IL-2-treated patients (67%) compared to control patients (33%) achieved increases of greater than 50% in the CD4+ T cell count (p = .08). Adverse events of grade 3 or grade 4 toxicity were infrequent in the current study and were substantially lower by comparison to those in studies of intermittent dose IL-2 therapy. Also, negligible changes in the HIV viral load from baseline to final measurement were observed in both groups. A trend toward a reduced number of modifications of antiretroviral therapy was apparent in the IL-2 group when compared to control patients. Conclusion: Daily, low-dose subcutaneous IL-2 therapy in conjunction with HAART is safe and well tolerated and is effective in expanding lymphocyte cell types including NK cells and naive T cells in individuals who have <300 CD4+ T cells.

Low-Dose Daily Subcutaneous Interleukin-2 in Combination with Highly Active Antiretroviral Therapy in HIV + Patients: A Randomized Controlled Trial

Purpose: Previous studies with intermittent interleukin-2 (IL-2) therapy using intermediate and high levels of IL-2 have demonstrated significant increases in the CD4 + T cell count in HIV-infected patients. Intermittent regimens are amenable to outpatient use, but severe adverse events are frequently experienced with intermediate- and high-dose levels of IL-2. Therefore in this study, the effect of daily, subcutaneous low-dose IL-2 therapy on safety and immunological endpoints was investigated to determine whether immunological benefit could be achieved without toxicity in HIV-infected patients also receiving highly active antiretroviral therapy (HAART). Method: A total of 115 patients were enrolled in the trial. Fifty-six asymptomatic HIV-infected patients who had CD4 + T cell counts less than 300 cells/μL at screening and a stable HIV viral load received low-dose IL-2 (1.2 million IU [MIU]/m 2 beginning dose) once daily in conjunction with HAART (IL-2 group). Fifty-nine patients received HAART alone (control group). Results: A dramatic effect of IL-2 on the natural killer (NK) cell population was observed with mean increases of 156 cells/μL in the IL-2 group compared to 19.93 cells/μL in the control group (p < .001). Additionally, IL-2-treated patients experienced a statistically significant increase in the mean percentage of CD4 + T cells (3.52% increase) when compared to control patients (1.33% increase) (p < .001). The expanded CD4 + T cell population was primarily of the naive phenotype, with mean increases of 4.53% for the IL-2 group and 0.31% for the control group (p < .001 for between-group difference). In addition, a higher proportion of IL-2-treated patients (67%) compared to control patients (33%) achieved increases of greater than 50% in the CD4+ T cell count (p = .08). Adverse events of grade 3 or grade 4 toxicity were infrequent in the current study and were substantially lower by comparison to those in studies of intermittent dose IL-2 therapy. Also, negligible changes in the HIV viral load from baseline to final measurement were observed in both groups. A trend toward a reduced number of modifications of antiretroviral therapy was apparent in the IL-2 group when compared to control patients. Conclusion: Daily, low-dose subcutaneous IL-2 therapy in conjunction with HAART is safe and well tolerated and is effective in expanding lymphocyte cell types including NK cells and naive T cells in individuals who have <300 CD4+ T cells.

Immune regulation in Cushing's syndrome: relationship to hypothalamic-pituitary-adrenal axis hormones.

Hormones of the hypothalamic-pituitary-adrenal (HPA) axis are connected closely with immune measures. To investigate whether Cushing's syndrome (CS) is associated with immune dysregulation, we compared the percentage of specific lymphocyte subsets as well as natural cell activity (NKCA) in 48 patients with Cushing's syndrome and 48 age- and sex-matched normal controls. Lymphocyte subset analysis included the percentage of lymphocytes expressing CD3 (total T), CD4 (T helper/inducer), CD8 (T suppressor/cytotoxic) and CD56 (NK cell) antigens. Baseline plasma concentrations of cortisol, ACTH and beta-endorphin as well as 24 h urinary-free cortisol (UFC) levels also were determined. Results indicated a decrease in the percentage of CD4+ cells (p < 0.05), an increase in percentage of CD8+ cells (p < 0.05), a decrease in CD4/CD8 ratios (p < 0.01), and a reduction in NKCA (p < 0.05) in patients with CS compared to matched controls. We also found significant negative correlations between NKCA on the one hand and 24 h UFC (p < 0.05) and plasma beta-endorphin (p < 0.05) on the other. These results indicate there is immune dysregulation in CS patients which can be explained in part by an increase in HPA-axis hormones.