Higher Percentage Of CD4 Research Articles

Abstract TP255: Lymphocytes And Platelets In Extracted Thrombus May Indicate The Etiology Of The Thrombus. Results From The Itacat Multicentric Registry.

Aim: We performed a histological and immune analysis of the clot in acute stroke patients to detect surrogates of stroke etiology. Methods: We conducted a prospective observational study of consecutive patients with acute stroke who underwent thrombectomy that obtained extracted thrombus (ITACAT study). Several staining were performed to evaluate red blood cells/fibrin (hematoxylin/eosin), platelets (CD61) and leucocytes (CD4, CD8 and CD20). All patients received CT angio to detect extra/intracranial vascular stenosis and 30-day cardiac monitoring to diagnose AF. According to TOAST classification the thrombi were classified in cardioembolic etiology CE (T-CE), due to symptomatic atherosclerosis (T-AT) and without any cause (T-CRYP). We excluded strokes due to double cause or incomplete workup. Results: Of the 117 patients: 30 were T-AT, 55 were T-CE and 32 were T-CRYP. T-AT patients were younger: T-AT 68 years (60-77) Vs. T-CE 75 years (68-80) Vs. T-CRYP 72 years (55-81) (p=0.034). T-AT group had higher percentage of CD4: T-AT 6.52% (4-13) Vs. T-CE (3.31% (12.9) Vs .T-CRYP 3.72% (1.5-12) (p=0.015) and lower percentage of CD61: T-AT 51.18% (34-68) Vs. CE 64.70% (19.56) Vs. CRYP 70.3% (19) (p=0.001). There were no correlation between CD4 and platelets. Both CD4 OR 1.05 (1-1.10) (p=0.020) and CD61 (OR 0.96 (0.94-0.98) (p=0.01) independently predicted T-AT from the age. Final analysis (n=400 cases) will be ended in September 2021. Conclusions: Patients with high percentage of CD4 and low percentage of CD61 are related to atherosclerosis etiology.

Anti‑VEGF antibody triggers the effect of anti‑PD‑L1 antibody in PD‑L1low and immune desert‑like mouse tumors.

The efficacy of programmed cell death-ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) blockade therapy has been demonstrated but is limited in patients with PD-L1low or immune desert tumors. This limitation can be overcome by combination therapies that include anti-vascular endothelial growth factor (VEGF) therapy. Such combinations have been investigated in clinical trials for a number of cancer types; however, evidence on the mechanisms underlying their effects in these types of patients is still not sufficient. Therefore, the present study investigated the efficacy and effects on CD8+ T cell and C-X-C motif chemokine receptor 3 (CXCR3) ligand expression in tumors by combining anti-PD-L1 and anti-VEGF antibodies using an OV2944-HM-1 mouse model with PD-L1low and immune desert-like phenotypes. Although the model exhibited anti-PD-L1 insensitivity, anti-PD-L1 antibody treatment combined with anti-VEGF antibody inhibited tumor growth compared with anti-VEGF monotherapy, which itself inhibited tumor growth compared with the control treatment on Day 25. In combination-treated mice, a higher percentage of CD8+ T cells and higher levels of CXCR3 ligands were observed in tumor tissues compared with those in the anti-VEGF antibody treatment group, which was not significantly different from control treatment on Day 8. The increase in the intratumoral percentage of CD8+ T cells following the combination treatment was reversed by CXCR3 blocking to the same level as the control. In an anti-PD-L1 insensitive model with PD-L1low and immune desert-like phenotypes, although anti-PD-L1 antibody alone was not effective, anti-PD-L1 antibody in combination with anti-VEGF antibody exhibited antitumor combination efficacy with an increase of CD8+ T cell infiltration, which was suggested to be dependent on the increase of intratumoral CXCR3 ligands. This mechanism could explain the efficacy of anti-PD-L1 antibody and anti-VEGF antibody combination therapy in the clinical setting.

Correlation of CD4+CD57+, CD8+CD57+, CD4+KLRG1+, CD8+KLRG1+ T Cells Percentage and Serum MMP-9 Level with Cognitive Dysfunction among Systemic Lupus Erythematosus Patients

Background: ‘Lupus brain fog’ is a phenomenon of cognitive function decline in SLE patients. Premature immunosenescence in SLE was presumed to play a significant role in the mechanism of cognitive dysfunction. Aim: To prove the correlation between the terminally-differentiated CD4+CD57+, CD8+CD57+, CD4+KLRG1+, and CD8+KLRG1+ T cells & serum levels of MMP-9 with cognitive dysfunction in SLE patients. Methods: 53 women SLE were conducted to perform MMSE and MoCA-Ina tests to evaluate cognitive function. Immunosenescence was observed by measuring the terminallydifferentiated CD4+CD57+, CD8+CD57+, CD4+KLRG1+, and CD8+KLRG1+ T cells, which were measured by flowcytometry. In addition, MMP-9, an enzyme produced by terminallydifferentiated T cells, was measured using ELISA. Results: SLE patients with cognitive dysfunction based on MMSE and MoCA-Ina test had higher percentage of CD4+CD57+, CD8+CD57+, CD4+KLRG1+, CD8+KLRG1+ T cells and serum levels of MMP-9 compared to patients with normal cognitive function. CD4+CD57+, CD8+CD57+, CD4+KLRG1+, CD8+KLRG1+ T cells percentage and serum MMP-9 level showed negative correlation with both MMSE scores (r = -0.286; r = -0.447; r = -0.279; r = -0.537; r = 0,411) and MoCA-Ina scores (r = -0.454; r = -0.539; r = -0.435; r = -0.535; r = -0.648). Meanwhile, percentage of CD4+CD57+, CD8+CD57+, CD4+KLRG1+ and CD8+KLRG1+ T cells showed positive correlation with serum MMP-9 level (r = 0.292; r = 0.414; r = 0.449; r = 0.374). Conclusion: Expansion of CD4+CD57+, CD8+C57+, CD4+KLRG1+, CD8+KLRG1+ terminally differentiated T cells & increase of serum MMP-9 level are correlated with cognitive dysfunction in SLE patients

Naturally Selected Anti-CD7 CAR-T Cells without Additional Genetic Manipulations As a Potentially Superior Therapy for T-Cell Malignancies

IntroductionTo prevent CAR-T fratricide, anti-CD7 CAR (7CAR) T cells used for treating T-cell malignancies are often modified by CD7 ablation via CRISPR/CAS9 gene editing or by co-expression of a CD7-specific protein expression blocker. Both methods require additional genetic manipulations of CAR-T. Here we transduce 7CAR into bulk T cells without CD7 disruption and thereafter allow CAR-T cells to emerge in vitro after fratricidal “natural selection”. The biological characteristics of these naturally selected anti-CD7 CAR (NS7CAR) T cells and their potential advantages in treating patients with T-cell malignancies are described.MethodsThe percentage of CD3 +CD7 - T cells in peripheral blood from either healthy donors (HDs) or patients (PTs) were determined by flow cytometry. Peripheral bulk T cells were positively selected using CD3 magnetic beads, and peripheral CD7 - T cells were negatively selected using CD7 magnetic beads. To avoid contamination from malignant T cells, patients only with CD3 -CD7 + T cell blasts were included in this study. The 7CAR gene cassette comprising of the cDNA of a CD7-specific antibody sequence fused to the coding sequences for the CD8TM-41BB-CD3z signal domains, and the T2A-linked tEGFR was cloned into a lentiviral vector backbone under the control of an EF1α promoter. 7CAR lentiviral transduction of bulk T cells (NS7CAR) or CD7 - T cells (Neg7CAR) were performed two days after CD3/CD28 dynabeads activation. CD7-ablated 7CAR T cells (KO7CAR) were derived by electroporation of bulk T cells with CD7-targeting Cas9-gRNA RNP 24 hours before 7CAR transduction. CAR-T cells were routinely kept in culture for 12 days. The levels of CD7 mRNA, protein, and surface expression were determined respectively by qualitative/quantitative reverse transcription PCR, Western blotting, and flow cytometry. Iv vitro cytotoxic activity for CD7 + tumor cell lines was tested using a flow-cytometry-based cytotoxicity assay. NSG mice engrafted with CCRF-CEM-luciferase cells were used as an animal model to validate the activities of CD7 CAR.ResultsThree approaches for generating anti-CD7 CAR-T cells were compared: NS7CAR (fratricidal natural selection from bulk T cells after 7CAR transduction), Neg7CAR (7CAR transduction of purified CD7-negative T cells) and KO7CAR (Cas9 RNP CD7 gene ablation). While CD7 - T cells were detectable in HDs of all ages (9.48±0.96%, n=13), we observed a significant increase of this cell population in T-cell acute lymphoblastic leukemia PTs (15.98±0.57%, n=13) (Fig 1A). We next tested the feasibility of using bulk T cells to generate naturally occurring 7CAR T cells without CD7 gene ablation or protein blockage. Three days after 7CAR lentiviral transduction, purified bulk peripheral T cells had a rapid and dramatic phenotypic transition from CD7 + CAR - to CD7 -CAR +(Fig 1B). Although fratricide led to a much lower expansion and viability of 7CAR T cells compared to T- cells without 7CAR transduction, approximately 80% of the 7CAR T cells were viable, making further studies feasible. After 12 days of culture, 7CAR T cells from PTs displayed stronger expansion potential (Fig 1C) and contained a larger CD8 + subpopulation (Fig 1D) as compared to cells derived from HDs. In comparison to Neg7CAR and KO7CAR, the final NS7CAR product displayed a lower expansion capability, but contained a higher percentage of CAR + cells, a larger CD8 +subset and an increased central memory phenotype (Table 2). Interestingly, although the final cells from all three products had no surface CD7 expression, mRNA and total protein were only detected from NS7CAR, but not from Neg7CAR or KO7CAR. Additionally, NS7CAR showed superior cytotoxicity and cytokine release in an in vitro functional test (Fig 1E). In the animal model, the NS7CAR conferred robust protection against leukemia progression with marked reduction in leukemia cell burden in the first two weeks after CAR T- cells injection (Fig 1F&G).ConclusionAmong the three approaches, the NS7CAR T cells was significantly enriched in CAR + cells and contained a higher percentage of CD8 + central memory T cells. Importantly, our data indicate that autologous PBMCs from patients were superior to PBMCs of healthy donors in yielding sufficient NS7CAR T cells for therapeutic needs. An investigator-initiated trial is currently ongoing to test the feasibility, efficacy, and safety of NS7CAR T cells for treating T-cell acute lymphoblastic leukemia. [Display omitted] DisclosuresLiu: SenlangBio: Current Employment. Ba: SenlangBio: Current Employment. Li: SenlangBio: Current holder of individual stocks in a privately-held company.

Role of B Cell Profile for Predicting Secondary Autoimmunity in Patients Treated With Alemtuzumab

ObjectiveTo explore if baseline blood lymphocyte profile could identify relapsing remitting multiple sclerosis (RRMS) patients at higher risk of developing secondary autoimmune adverse events (AIAEs) after alemtuzumab treatment.MethodsMulticenter prospective study including 57 RRMS patients treated with alemtuzumab followed for 3.25 [3.5-4.21] years, (median [interquartile range]). Blood samples were collected at baseline, and leukocyte subsets determined by flow cytometry. We had additional samples one year after the first cycle of alemtuzumab treatment in 39 cases.ResultsTwenty-two patients (38.6%) developed AIAEs during follow-up. They had higher B-cell percentages at baseline (p=0.0014), being differences mainly due to plasmablasts/plasma cells (PB/PC, p=0.0011). Those with no AIAEs had higher percentages of CD4+ T cells (p=0.013), mainly due to terminally differentiated (TD) (p=0.034) and effector memory (EM) (p=0.031) phenotypes. AIAEs- patients also showed higher values of TNF-alpha-producing CD8+ T cells (p=0.029). The percentage of PB/PC was the best variable to differentiate both groups of patients. Baseline values >0.10% closely associated with higher AIAE risk (Odds ratio [OR]: 5.91, 95% CI: 1.83-19.10, p=0.004). When excluding the 12 patients with natalizumab, which decreases blood PB/PC percentages, being the last treatment before alemtuzumab, baseline PB/PC >0.1% even predicted more accurately the risk of AIAEs (OR: 11.67, 95% CI: 2.62-51.89, p=0.0007). The AIAEs+ group continued having high percentages of PB/PC after a year of alemtuzumab treatment (p=0.0058).ConclusionsA PB/PC percentage <0.1% at baseline identifies MS patients at low risk of secondary autoimmunity during alemtuzumab treatment.​

Microparticles and PD1 interplay added a prognostic impact in treatment outcomes of patients with multiple myeloma

Although multiple myeloma (MM) is still considered as an incurable disease by current standards, the development of several combination therapies, and immunotherapy approaches has raised the hope towards transforming MM into an indolent, chronic disease, and possibly achieving a cure. We tried to shed light on the expression of PD1 and different Microparticles (MPs) in MM and their interplay as a mechanism of resistance to standardized treatments, in addition, find their associations with prognostic factors of symptomatic MM. Thirty patients with newly diagnosed and chemotherapy naïve active MM, along with 19 healthy participants of comparable age and sex were recruited, after diagnosis of MM; blood samples were collected from both patients and controls for flow cytometric detection of CD4+, CD8+, CD4+PD1+, and CD8+PD1+T cells, total MPs, CD138+ MPs, and platelet MPs. MM patients had statistically significant higher levels of TMPs, CD138+ MPs compared to their controls, while PMPs exhibited no significant difference between both groups. Statistically significant higher percentages of CD8+, PD1CD8+, PD1CD4+T cells were detected in patients compared to controls, while the latter group had a significantly higher percentage of CD4+T cells than MM patients, patients who did not achieve complete response, had significantly higher percentages of PMPs, CD138+MPs, PD1+CD8+, PD1+CD4+, and CD8+T cells (cutoff values = 61, 10.6, 13.5, 11.3 and 20.1 respectively), (p-values = 0.002, 0.003, 0.017, 0.001 and 0.008 respectively). Microparticles and PD1 expressions were associated with proliferative potential and resistance to Bortezomib-based treatments, our results suggested that they played a crucial role in myeloma progression.

Temporal Dynamics of T Helper Populations in the Proximal Small Intestine after Oral Bovine Lactoferrin Administration in BALB/c Mice.

Bovine lactoferrin (bLf), a component of milk and a dietary supplement, modulates intestinal immunity at effector and inductor sites. Considering the regional difference in intestinal compartments and the dynamics of local cytokine-producing cells in the gut across time, the aim of this work was to characterize the effects of bLf on the proximal small intestine in a BALB/c murine model of oral administration. Male BALB/c mice were treated with oral bLf vs. saline control as mock by buccal deposition for 28 days. Intestinal secretions were obtained at different time points and cells were isolated from Peyer’s patches (PP) and lamina propria (LP) of the proximal small intestine as representative inductor and effector sites, respectively. Total and specific anti-bLF IgA and IgM were determined by enzyme-immuno assay; the percentages of IgA+ and IgM+ plasma cells (PC) and cytokine-producing CD4+ T cells of PP and LP were analyzed by flow cytometry. We found that total and bLf-specific IgA and IgM levels were increased in the intestinal secretions of the bLf group in comparison to mock group and day 0. LP IgA+ PC and IgM+ PC presented an initial elevation on day 7 and day 21, respectively, followed by a decrease on day 28 in comparison to mock. Higher percentages of CD4+ T cells in LP were found in the bLf group. Cytokines-producing CD4+ T cells populations presented a pattern of increases and decreases in the bLf group in both LP and PP. Transforming growth factor beta (TGF-β)+ CD4+ T cells showed higher percentages after bLf administration with a marked peak at day 21 in both LP and PP in comparison to mock-treated mice. Oral bLf exhibits complex immune properties in the proximal small intestine, where temporal monitoring of the inductor and effector compartments reveals patterns of rises and falls of different cell populations. Exceptionally, TGF-β+ CD4+ T cells show consistent higher numbers after bLf intervention across time. Our work suggests that isolated measurements do not show the complete picture of the modulatory effects of oral bLf in immunological sites as dynamic as the proximal small intestine.

Analysis of characteristics and predictive factors of immune checkpoint inhibitor-related adverse events.

Objective:We aimed to retrospectively analyze the toxicity profiles and predictors of immune-related adverse events (irAEs) as well as the correlation between irAEs and the clinical efficacy of multi-type immune checkpoint inhibitors (ICIs) in patients with advanced pan-cancer in a real-world setting.Methods:We retrospectively analyzed data from 105 patients with advanced pan-cancer treated with multi-type ICIs at the First Hospital of Jilin University between January 1, 2016 and August 1, 2020. We used logistic regression analyses to investigate the associations of irAEs with clinical baseline characteristics, blood count parameters, and biochemical indicators during treatment. Receiver operating characteristic curves were used to determine cutoff values for parameters and area under the curve values. Kaplan–Meier and Cox multivariate regression analyses were performed to estimate the relationships of baseline characteristics and irAEs with progression-free survival (PFS) and overall survival (OS).Results:A lower relative lymphocyte count (cutoff = 28.5%), higher albumin level (cutoff = 39.05 g/L), and higher absolute eosinophil count (AEC) (cutoff = 0.175 × 109/L) were significantly associated with the occurrence of irAEs, among which a higher AEC (cutoff = 0.205 × 109/L) was strongly associated with skin-related irAEs [odds ratios (ORs) = 0.163, P = 0.004]. Moreover, a higher lactate dehydrogenase level (cutoff = 237.5 U/L) was an independent predictor of irAEs of grade ≥ 3 (OR = 0.083, P = 0.023). In immune cell subgroup analysis, a lower absolute count of CD8+CD28− suppressor T cells (OR = 0.806; 95% confidence interval: 0.643–1.011; P = 0.062), which are regulatory T lymphocytes, was associated with the occurrence of irAEs, although the difference was not statistically significant. Furthermore, a higher percentage of CD19+ B cells was associated with the occurrence of irAEs of grade ≥ 3 (P = 0.02) and grade ≥ 2 (P = 0.051). In addition, patients with any grade of irAE had a significantly high PFS (8.37 vs. 3.77 months, hazard ratios (HR) = 2.02, P = 0.0038) and OS (24.77 vs. 13.83 months, HR = 1.84; P = 0.024).Conclusions:This retrospective study reports clinical profile data for irAEs in unselected patients in a real-world setting and explored some parameters that may be potential predictive markers of the occurrence, type, or grade of irAEs in clinical practice. Evidence of a correlation between safety and efficacy may facilitate a complete assessment of the risk-benefit ratio for patients treated with ICIs.

Peripheral Blood As a Substitute for Bone Marrow for Assessing the Immune Profile in AML

Background: Immunotherapy has become a major strategy for cancer therapy and has dramatically improved patients' outcomes, especially in hematologic malignancies, but little is known about the functional capacity of T cells in Acute Myeloid Leukemia (AML) patients. The AML Bone Marrow (BM) microenvironment is a dynamic structure that plays a crucial role in initiating and maintaining leukemogenesis and contributing to AML drug resistance and immune suppression, and it is likely that T cells extant in the AML environment are functionally impaired. For example, pre-clinical and clinical studies have identified the upregulation of PD-1/PD-L1 axis as a potential mechanism of resistance to T-cell therapy suggesting that a better understanding of the expression of immune regulatory molecules in AML will help guide future therapeutic interventions, as well as serve as potential predictive/prognostic biomarkers for T-cell therapies. Frequent bone marrow sampling is not feasible in AML patients and so it is important to understand whether peripheral blood T-cells reflect T-cell phenotypes in the BM, including expression of inhibitory molecules. The objective of this study is to evaluate the use of Peripheral Blood (PB) as a surrogate for BM to support periodic immunophenotype monitoring in a clinical trial setting.Methods: Patient matched cryopreserved BM and PB primary AML samples (n=10 pairs) were surveyed by multiparameter flow cytometry to quantitate parameters including CD4 and CD8 effector T cell populations (Naïve: CD45RAhiCCR7hi, Central Memory: CD45RAloCCR7hi, Effector Memory: CD45RAloCCR7lo, Terminal Effector CD45RAhiCCR7lo), regulatory T cells (CD4+CD25+CD127-), expression of various immune checkpoint molecules (PD-1, TIM3, LAG3 and TIGIT), and leukemia cell phenotypes such as CD33, CD34, CD38, PD-1 and TIGIT ligands. All parameters collected for T cells and AML cells were analyzed using the Bland-Altman and Lin's concordance methods to evaluate the similarities between BM and PB. For eight samples, primary AML mononuclear cells (MNCs) were treated ex vivo for 4 days with Control or AMG 330 (CD33/CD3 BiTE®) and phenotyped to monitor response to BiTE® treatment and changes in checkpoint receptors.Results: PB is an appropriate surrogate for BM for many of the T cell and AML cell attributes analyzed. CD33 expression on blasts and the distribution of CD4 effector subsets in PB and BM were concordant based on Lin's concordance correlation, while CD4:CD8 ratio and T cell:blasts ratio were qualitatively similar (Spearman Rank). We also observed that expression of CD25 on CD4 and CD8 T cells and the AML progenitor population CD34+/CD38+ in PB and BM were linearly correlated. CD69 was consistently expressed on a higher percentage of CD4 and CD8 T cells in the BM compared to PB, consistent with its role in memory T cell retention in the BM. Checkpoint receptor expression was similar PB and BM (see Figure 1) but did not meet the statistical test for concordance; analysis of a larger number of samples will provide greater insights into non-correlated samples. We observed that most of the AML samples had a high median frequency of T cells expressing PD-1 (CD4+: 36%; CD8+: 46%) and TIGIT (CD4+: 22%; CD8+: 52%). TIM3 expression was lower (CD4+: 18%; CD8+: 17%) while LAG3 was rarely expressed on AML T cells (2/9 AML samples tested had >5% LAG3+). We also observed that PD-1 was expressed more frequently on CD8+ T cells from R/R AML BM compared to ND (medians: 43% vs 53%, respectively, p=0.022). TIGIT ligands, PVR and PVRL2, were present on AML blasts in the majority of patients (7/9 and 5/9 had >20% PVR+ or PVRL2+ respectively), while PD-L1 and PD-L2 were not expressed on blasts in any of the AML samples tested. To understand if these immunophenotypes relate to BiTE® responsiveness, primary AML MNCs (n=9) were treated ex vivo for 4 days with AMG 330. Response of these samples to treatment was dependent on CD33 expression and T cell:target cell ratio. Changes in PD-1 and TIGIT induced by ex vivo AMG 330 treatment will also be presented.Conclusion: Using multiparameter flow analysis, we observed a strong correlation between BM and PB for several biomarkers tested including CD4:CD8 ratios, T cell effector subsets, and leukemia cell phenotypic markers. Our results suggest that PB immunophenotype is representative of the BM in AML and can be used in a clinical trial setting to gain relevant biomarker data. [Display omitted] DisclosuresLesley:Amgen Inc.: Employment, Equity Ownership. Beutner:Amgen Inc.: Employment, Equity Ownership. Anderson:Amgen Inc.: Employment, Equity Ownership. Fitzpatrick:Amgen Inc.: Employment, Equity Ownership. Loberg:Amgen Inc.: Employment, Equity Ownership. Mehta:Amgen Inc.: Employment, Equity Ownership. Dos Santos:Amgen Inc.: Employment.

Effect of Azacitidine on CD4+CD25+Foxp3+ Regulatory T Cell on Immunity in Myelodysplastic Syndrome Survivors

Background: Immune editing is recognized as important in various pathogenic processes. We previously showed that regulatory T cell (Treg) levels were elevated in MDS with a poor prognosis and that Treg levels decreased with Azacitidine (AZA) treatment and became equivalent to that in the untreated group. In this study, we examined changes in the number of circulating Tregs, T cells, and NK cells in the peripheral blood of patients with MDS treated with AZA, and we evaluated the clinical significance of these findings with survival.Patients and Methods: Patients with MDS and with acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) according to the WHO 2008 classification who were initially treated between January 2015 and December 2016 and followed up for more than 6 months were enrolled in the study. AZA was given intravenously at a dose of 75 mg/m2 daily for either 5 or 7 consecutive days every 4 weeks. The response was assessed using the IWG response criteria for MDS. A total of 50 patients with AML-MRC (n=11), refractory anemia with excess blasts (RAEB) (n=8), refractory cytopenia with multilineage dysplasia (RCMD) (n=28), myelodysplastic/myeloproliferative neoplasms (MDS/MPN) (n=12), and refractory anemia with ring sideroblastis (RARS) (n=1) were included in the study. The IPSS was classified as low (n=15) intermediate-1 (n=13), intermediate-2 (n=10), and high (n=13). Twenty-six patients did not require treatment, including a transfusion (untreated group). Twenty-two patients were treated with AZA (AZA group). The remaining patients received conventional chemotherapy. After obtaining informed consent, blood was drawn from all of the patients at initial diagnosis. In the 22 patients treated with AZA, blood was also drawn after 6 completed treatment cycles. CD4+CD25+Foxp3+ Treg cells, CD3+CD4+ T cells, CD3+CD8+ T cells, and CD3-CD56+ NK cells were analyzed using flow cytometry, and the absolute numbers of these cells were calculated. Overall survival (OS) probabilities were estimated using the Kaplan-Meier method and compared by the log-rank test for univariate analysis.Results: The median age was 74 years (range: 44-93 years), and 35 of the patients were male. The median period of observation was 391 days. In the AZA group, 10 patients died (AZA-ineffective group), and 12 had either a CR, PR, or HI (AZA-effective group). The 2-year OS of the AZA group was 43.7%. The absolute number of Tregs and Treg% values before treatment were not significantly different between the AZA group and the untreated group. The Treg levels in peripheral blood before treatment in the AZA-effective (0.80±0.39%) tended to be lower than that in the AZA-ineffective (1.29±0.82%) group (p=0.076). The CD8+ T cell counts before treatment were moderately higher in the AZA-effective group compared to that in the AZA-ineffective group (385.5 ± 129.0 vs. 195.5 ± 70.0 /μL, p<0.01). Similarly, the CD3-CD56+ NK cell counts before treatment differed significantly between the AZA-effective group (197.4 ± 138.8 /μL) and the AZA-ineffective group (82.3 ± 36.7 /μL) (p=0.015). In a univariate analysis, the MDS patients with elevated NK cells survived longer than the group with reduced NK cells (p=0.013). In a multivariate analysis, the lower percentage of Treg cells and the higher percentage of CD8+T cells before treatment were independent factors for OS (HR, 4.80; p=0.085, HR, 4.13; p=0.040, respectively). In the AZA group, the percentage of CD8+T cells before treatment associated with OS (HR, 39.24; p=0.018). After treatment, the AZA group had an increase in the NK cell counts, which tended to associate with survival (HR 4.29; p=0.067).Conclusions: The number of Tregs in MDS cases requiring treatment was higher than that in MDS cases for which treatment was unnecessary. The prognosis of the patients with high CD8+ T cells counts was better. In contrast, there was an increase in the number of NK cells after AZA treatment, which was found to be associated with an improved prognosis. These results suggest that an immunological mechanism associated with the prognosis of MDS may be enhanced by treatment with AZA. Further examination of this mechanism and of the influence of AZA on the immunological effect are required. DisclosuresNo relevant conflicts of interest to declare.

Integrated Analysis of Genomic and Immunological Features in Lung Adenocarcinoma With Micropapillary Component.

BackgroundMicropapillary adenocarcinoma is one of the most aggressive histologic subtypes of lung adenocarcinoma (LADC), and even a minor proportion of micropapillary component (MPC) within the LADC could contribute to poor prognosis. Comprehensive analysis of genetic and immunological features of LADC with different percentages of MPC would help better understand cancer biology of this LADC subtype and direct future treatments.MethodsWe performed next-generation sequencing (NGS) for a discovery cohort of 43 LADC patients whose tumors were micro-dissected to separate MPC and non-MPC lesions and a reference cohort of 113 LADC patients. MPC-enriched genetic alterations that were detected in the discovery cohort were then confirmed using a validation cohort of 183 LADC patients. Immunological staining was also conducted on the MPC-containing samples in the discovery cohort.ResultsTumors with a higher percentage of MPC tended to harbor more tumor mutation burdens (TMBs) and chromosome instability (CIN). Some rare genetic events may serve as the genetic landscape to drive micropapillary tumor progression. Specifically, alterations in transcription termination factor 1 (TTF1), brain-specific angiogenesis inhibitor 3 (BAI3), mammalian target of rapamycin (MTOR), and cyclin-dependent kinase inhibitor 2A (CDKN2A) were cross-validated to be enriched in MPC-contained LADC. Additionally, tumors with a higher percentage of MPC were associated with a higher percentage of CD4+, CD8+, and PD-L1+ staining, and some genetic changes that were enriched in MPC, including MET amplification and MTOR mutation, were correlated with increased PD-L1 expression.ConclusionWe identified multiple novel MPC-enriched genetic changes that could help us understand the nature of this aggressive cancer subtype. High MPC tumors tended to have elevated levels of TMBs, T cell infiltration, and immunosuppression than low MPC tumors, implying the potential link between MPC content and sensitivity to immunotherapy.

Cytokine production in ex-vivo stimulated fresh and cryopreserved T-cells

Abstract Objective: In vitro cytokine production by peripheral blood mononuclear cells (PBMCs) is an important and reliable measure of immunocompetence. PBMC can be stimulated directly after isolation or frozen for later use. However, cryopreservation may affect cell recovery, viability and functionality. This study aims to investigate cytokine synthesis in ex-vivo stimulated fresh and cryopreserved CD4+ and CD4- T cells. Methods: PBMCs were obtained by Ficoll gradient centrifugation from heparinized peripheral blood of 6 middle-aged clinically healthy subjects. Half of these cells (labeled “Fresh”) was further processed and the other half (labeled “Cryo”) was cryopreserved at -140°C for up to 3 months. Fresh-PBMCs were activated with Phorbol-Myristate-Acetate/Ionomycin/Monensin for 5 hours immediately after isolation while Cryo-PBMCs were identically activated after thawing and cell resting. Activated cells were fixed, permeabilized and intracellular cytokine staining was performed using Phycoerythrin (PE)-conjugated antibodies for Interleukin-2 (IL-2), Tumor Necrosis Factor-alpha (TNF-a), and Interferon-gamma (IFN-g). All samples were analyzed within 24 hours by flow cytometry. Results: Both Fresh and Cryo CD3+CD4+/CD3+CD4- sub-populations partially produced each of the three cytokines. A higher percentage of CD4+ T cells produced IL-2 and TNF-a and a greater percentage of CD4- T cells were found to produce IFN-g. A significantly higher percentage of Cryo-lymphocytes was shown to produce TNF-a in both CD3+CD4+ (31.4% vs 24.9%, p=0.031) and CD3+CD4- (22.7% vs 17.9%, p=0.031) subpopulations. No notable difference was found for IL-2 and IFN-g production between Fresh and Cryo T cells. Conclusion: Cryopreservation for up to 3 months significantly increases TNF-a production of T-cells in clinically healthy middle-aged subjects.

Temporal and spatial characterization of negative regulatory T cells in HIV-infected/AIDS patients raises new diagnostic markers and therapeutic strategies.

BackgroundNegative regulatory T cells (Tregs) not only deplete effector T cells but also inhibit the clearance of HIV during infection, which may allow Tregs to be used as informative diagnostic markers. To facilitate both diagnosis and treatment, a thorough understanding of these regulators by characterizing them on temporal and spatial scales is strongly required.MethodsHundred HIV‐infected/AIDS patients, including 87 males, with an average age of 35.8 years, as well as 20 healthy controls, were enrolled. Flow cytometry was used to analyze CD3+T cells, CD4+T cells, and CD8+T cells to evaluate the immune status of the participants. Then, a group of representative negative regulatory T cells, including CD4+PD‐1+T cells, CD4+PD‐1highT cells, CD8+PD‐1+T cells, and CD4+CD25high Tregs was also analyzed to explore their effects on disease progression and intercorrelation.ResultsThe percentages of CD4+PD‐1+T cells and CD4+CD25highTregs increased in patients with the same ultrahigh significance. Temporally, the patients with both intermediate‐stage and late‐stage disease had higher percentages of CD4+PD‐1+T cells; however, the percentage of CD4+CD25highTregs only increased in the patients with late‐stage disease. In addition, CD4+PD‐1+T cells but not CD4+CD25highTregs were negatively correlated with the absolute CD4+T cell count. Spatially, no correlations between CD4+PD‐1+T cells and CD4+CD25highTregs were observed, which suggests these Tregs function differently during immunosuppression.ConclusionsThis study characterized negative regulatory T cells in HIV‐infected/AIDS patients at both temporal and spatial scales and found that CD4+CD25+Tregs and CD4+PD‐1+T cells could be used as potential diagnostic markers for identifying different disease stages and monitoring disease progression.

Flow cytometric analysis of T lymphocytes and cytokines in aqueous humor of patients with varicella zoster virus-mediated acute retinal necrosis

BackgroundThe purpose of this study is to investigate the aqueous humor (AH) T lymphocyte subsets and cytokines of acute retinal necrosis (ARN) to elucidate the immunologic inflammatory features of this disorder.MethodsThree patients with ARN infected with varicella zoster virus (VZV) who underwent multiple intravitreal injections of ganciclovir were enrolled in this study. The control group consisted of four non-infectious patients with acute anterior uveitis (AAU). Flow cytometric analysis was performed on the lymphocyte subsets from the AH and peripheral blood (PB) samples during the active phase of intraocular inflammation. Five inflammatory cytokines were measured in each AH sample and various clinical characteristics were also assessed.ResultsVZV deoxyribonucleic acid (DNA) was detected by real-time polymerase chain reaction (PCR) in AH from all the ARN patients, who showed higher CD8+ T lymphocytes population in AH than the AAU patients (p = 0.006). CD4/CD8 ratios of T lymphocytes and the percentage of CD8 + CD25+ T lymphocytes in AH were significantly lower in ARN than in AAU (p = 0.006; p = 0.012). In the ARN patients, the percentages of CD4+ and CD8+ T lymphocytes in AH were higher than those found in PB. The percentage of CD4 + CD25+ T lymphocytes in AH was significantly higher than the proportion in PB in the AAU patients (p = 0.001). Immunoregulatory cytokine Interleukin-10 in AH was significantly elevated in the ARN patients in comparison with the case of the AAU patients (p = 0.036). In ARN, the copy number of VZV DNA in AH positively correlated with the percentage of CD8+ T lymphocytes in AH and negatively correlated with the CD4/CD8 ratio in AH during the course of disease treatment (p = 0.009, r = 0.92; p = 0.039, r = − 0.834).ConclusionThe ARN patients caused by VZV had different intraocular T lymphocyte subsets and cytokines profile than those of the non-infectious patients. High percentages of CD8+ T lymphocytes and low CD4/CD8 T cell ratios may be a potential biomarker for diagnosis of viral-infectious uveitis. T lymphocytes examination at the inflammatory sites has the potential to become a useful research tool for differentiating viral and non-viral uveitis.

AT2 Receptor Activation Induces Treg Cells and Mitigates Renal Ischemic Injury

Kidney infiltrating immune cells such as monocytes, neutrophils, and T-cells play critical roles in renal ischemia-reperfusion (IR) injury and repair. Recently, angiotensin AT2R has been implicated in protecting kidney against injury and monocytes infiltration, particularly in chronic kidney disease. However, the role of AT2R in IR injury and repair phases, and T-cell modulation is unknown. To address this question, Sprague-Dawley rats were administered with the AT2R agonist C21 (0.3 mg/kg, i.p.) two hour before 30-minute bilateral renal ischemia. IR caused an early (2h post-ischemia) renal functional injury as indicated by elevated proteinuria, blood urea nitrogen and plasma creatinine, and enhanced immune cells (T-cells and CD4 T-cells) infiltration and levels of the proinflammatory cytokines TNF-α and IL-6. The C21 treatment reversed these changes and increased the anti-inflammatory IL-10 level. During the repair phase at day 3 and 5, renal functional injury and T cells infiltration in IR and IR+C21 returned to normal as sham controls. However, high percentages of CD4+FoxP3+ (Tregs, known for their role in repair), and CD4+IL-10+ cells in the kidney were observed in IR+C21 group compared with IR control. These data indicate that the AT2R activation protects the kidney against IR injury and immune cell infiltration in the early phase and modulates CD4 T-cells towards Tregs phenotype, which may have long-term beneficial effects on kidney function.

Effect of chemoradiotherapy on the proportion of circulating lymphocyte subsets in patients with limited-stage small cell lung cancer.

Chemoradiotherapy (CRT) is the standard treatment for limited-stage small cell lung cancer (LS-SCLC), which can exert anti-tumor effects by regulating immune cells. Different immune cell subsets are associated with a specific sensitivity to CRT. The purpose of this study was to characterize the proportion or composition of peripherallymphocytes in patients with LS-SCLC before and after CRT, and evaluate their prognostic value. A total of 98 patients with LS-SCLC were enrolled. The expression of CD3, CD4, CD8, CD45RA, CD45RO, CD38, CD56, and CD19 on the surface of peripheral blood cells was detected by flow cytometry and retrospectively analyzed. The relationship between the proportion of lymphocyte subsets, progression-free survival (PFS), and overall survival (OS) was evaluated using a log-rank test and Cox regression model. The median PFS was 12.3months and the median OS was 21.7months. Compared with the pre-treatment specimens, post-treatment lymphocytes had increased proportions of CD3+, CD3+CD8+, CD8+CD38+ T cells, and NKT cells, and a decreased proportion of CD3+CD4+ T cells, CD4+CD45RA+ T cells, B cells, NK cells, and CD4/CD8 ratio. Univariate and multivariate analyses showed that prophylactic cranial irradiation, high percentages of CD4+CD45RA+, CD8+CD38+ T cells after CRT independently predicted superior PFS. Male patients with a high baseline CD4+CD45RO+ T cell ratio predicted a poor OS. CRT induced changes in the proportion of circulating lymphocyte subsets in LS-SCLC, which is helpful for designing a regimen of immune drugs to be combined with CRT. The prognostic value of the proportion of lymphocytes aids in understanding the role of peripheral immune profiles in LS-SCLC.

Immune classification of clear cell renal cell carcinoma

Since the outcome of treatments, particularly immunotherapeutic interventions, depends on the tumor immune micro-environment (TIM), several experimental and computational tools such as flow cytometry, immunohistochemistry, and digital cytometry have been developed and utilized to classify TIM variations. In this project, we identify immune pattern of clear cell renal cell carcinomas (ccRCC) by estimating the percentage of each immune cell type in 526 renal tumors using the new powerful technique of digital cytometry. The results, which are in agreement with the results of a large-scale mass cytometry analysis, show that the most frequent immune cell types in ccRCC tumors are CD8+ T-cells, macrophages, and CD4+ T-cells. Saliently, unsupervised clustering of ccRCC primary tumors based on their relative number of immune cells indicates the existence of four distinct groups of ccRCC tumors. Tumors in the first group consist of approximately the same numbers of macrophages and CD8+ T-cells and and a slightly smaller number of CD4+ T cells than CD8+ T cells, while tumors in the second group have a significantly high number of macrophages compared to any other immune cell type (P-value <0.01). The third group of ccRCC tumors have a significantly higher number of CD8+ T-cells than any other immune cell type (P-value <0.01), while tumors in the group 4 have approximately the same numbers of macrophages and CD4+ T-cells and a significantly smaller number of CD8+ T-cells than CD4+ T-cells (P-value <0.01). Moreover, there is a high positive correlation between the expression levels of IFNG and PDCD1 and the percentage of CD8+ T-cells, and higher stage and grade of tumors have a substantially higher percentage of CD8+ T-cells. Furthermore, the primary tumors of patients, who are tumor free at the last time of follow up, have a significantly higher percentage of mast cells (P-value <0.01) compared to the patients with tumors for all groups of tumors except group 3.

Construction and Immunogenicity Comparison of Three Virus-Like Particles Carrying Different Combinations of Structural Proteins of Avian Coronavirus Infectious Bronchitis Virus.

Infectious bronchitis virus (IBV) poses massive economic losses in the global poultry industry. Here, we firstly report the construction and immunogenicity comparison of virus-like particles (VLPs) carrying the S, M and E proteins (SME-VLPs); VLPs carrying the S and M proteins (SM-VLPs); and VLPs carrying the M and E proteins (ME-VLPs) from the dominant serotype representative strain GX-YL5 in China. The neutralizing antibody response induced by the SME-VLPs was similar to that induced by the inactivated oil vaccine (OEV) of GX-YL5, and higher than those induced by the SM-VLPs, ME-VLPs and commercial live vaccine H120. More importantly, the SME-VLPs elicited higher percentages of CD4+ and CD8+ T lymphocytes than the SM-VLPs, ME-VLPs and OEV of GX-YL5. Compared with the OEV of GX-YL5, higher levels of IL-4 and IFN-γ were also induced by the SME-VLPs. Moreover, the mucosal immune response (sIgA) induced by the SME-VLPs in the tear and oral swabs was comparable to that induced by the H120 vaccine and higher than that induced by the OEV of GX-YL5. In the challenge experiment, the SME-VLPs resulted in significantly lower viral RNA levels in the trachea and higher protection scores than the OEV of GX-YL5 and H120 vaccines, and induced comparable viral RNA levels in the kidneys, and tear and oral swabs to the OEV of GX-YL5. In summary, among the three VLPs, the SME-VLPs carrying the S, M and E proteins of IBV could stimulate the strongest humoral, cellular and mucosal immune responses and provide effective protection, indicating that it would be an attractive vaccine candidate for IB.

Biomarkers associated with blinatumomab outcomes in acute lymphoblastic leukemia

This study aimed to identify biomarkers for clinical outcomes in a phase 3 clinical study of blinatumomab or chemotherapy in adults with Philadelphia chromosome-negative relapsed/refractory B-cell precursor acute lymphoblastic leukemia. Patients were randomized 2:1 to receive blinatumomab, a BiTE® therapy, for 4 weeks (9 μg/day cycle 1 week 1, 28 μg/day thereafter) every 6 weeks, or chemotherapy. Baseline blood samples were evaluated to identify biomarkers prognostic (both treatment groups) or predictive (either treatment groups) for overall survival, event-free survival, hematologic remission, minimal residual disease (MRD) response, duration of response, or adverse events. Baseline values were balanced between treatment groups. Prognostic biomarkers were platelets, tumor burden, and percentage of T cells: each 1-log increase in platelets at baseline was prognostic for improved 6-month survival; lower tumor burden was prognostic for hematologic remission; and a higher percentage of CD3+ T-cells was prognostic for MRD response. Consistent with the BiTE mechanism of action, higher percentage of CD45+ CD3+ CD8+ T cells was associated with hematologic remission following blinatumomab. No examined biomarkers were significant for the risk of grade ≥3 adverse events. Incorporating baseline biomarkers into future studies may help to identify subgroups most likely to benefit from blinatumomab.

The Predict Significance of ALDH Activity to the Relapse of t(8;21) Acute Myeloid Leukemia Patients at Complete Remission

To investigate the predict significance of the high aldehyde dehydrogenase activity (ALDH+) propitiation to the relapse of t(8;21) acute myeloid leukemia(AML) patients before and after treatment. Bone marrow samples of 23 t(8;21) AML patients diagnosis and achieved complete remission in our hospital from April 2015 to June 2016 were collected, then flow cytometry method was used to detect the activity of ALDH, relationship between it and relapse was analyzed. All the patients were followed up for a median of 32 (2-52) months. The median percentage of CD34+CD38- and CD34+CD38-ALDH+ cells among nucleated cells were 2.7 (0.36-35.7)% and 0.017 (0.0013-0.62)% at diagnosis, respectively. Among the bone marrow samples in patients achieved CR, the median percentage of CD34+CD38- cells was 0.035 (0.0064-0.66)%, and it was significantly decreased as compared with the percentage at diagnosis (P<0.001); The median percentage of CD34+CD38-ALDH+ cells was 0.014 (0.0019-0.24)%, and it showed no different as compared with the percentage at diagnosis (P=0.45). Survival analysis showed that patients with higher percentage of CD34+CD38- and CD34+CD38-ALDH+ cells at diagnosis tended to the lower 3-year relapse-free survival (RFS) (P=0.27 and 0.21). Furthermore, patients with higher percentage of CD34+CD38- and CD34+CD38-ALDH+ cells when achieved CR had a significant lower 3-year RFS rates (P=0.010 and 0.0044) as compared with those with lower percentage of CD34+CD38- and CD34+CD38-ALDH+ cells. Multivariate analysis showed that higher percentage of CD34+CD38-ALDH+ cells when achieved CR was an independent factor affecting RFS of the patients. The percentage of CD34+CD38-ALDH+ cells at CR in t(8;21) AML patients could predicts relapse, and had more profound predictive significance for relapse than the percentage of CD34+CD38- cells.