scholarly journals THU0104 THE GUT MICROBIOTA AND ITS RELEVANCE TO PERIPHERAL T REGULATORY CELLS AND T HELPER 17 IN PATIENTS WITH RHEUMATOID ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 266.1-266
Author(s):  
Y. LI ◽  
S. X. Zhang ◽  
X. F. Yin ◽  
Z. Mingxing ◽  
J. Luo ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
Gut Microbiota ◽  
Lymphocyte Subsets ◽  
Joint Destruction ◽  
Absolute Number ◽  
P Value ◽  
Rrna Gene ◽  
Healthy Controls ◽  
The Absolute

Background:Rheumatoid arthritis (RA) is a common autoimmune disorder with joint destruction and synovial inflammation characterized by abnormal immune responses to autoantigens. Our previous studies have demonstrated that impaired peripheral lymphocytes especially insufficiency of regulatory T cells (Tregs) played an important role in pathogenesis of RA1 2. Interestingly, the dysbiosis of gut microbiota triggers several types of autoimmune diseases through the imbalance of T lymphocyte subsets3. However, the detailed gut microbiota of RA patients and its correlation with Tregs and helper T cells 17 (Th17) are unclear up until now.Objectives:To compare the difference of gut microbiota between RA and healthy controls (HCs), and to investigate the relevance of gut microbiota with circulating Tregs and Th17 in patients with RA.Methods:From December 2018 to August 2019, a total of 205 diagnosed patients with RA and 199 age and sex-matched HCs were enrolled in this study. Stool of Every participant was collected for bacterial DNA extraction and 16S ribosomal RNA (rRNA) gene sequencing. The absolute numbers of Tregs and Th17 in PB of these individuals were measured by Flow Cytometer (FCM) combined with standard absolute counting beads. Data were expressed as mean ± standard deviation to the distribution. Independent-samples T test and Spearman rank correlation test. P value <0.05 were considered statistically significant.Results:Patients with RA had a significantly difference of diversity and abundance of intestinal microbiota compared with those of HCs (P< 0.05). Detailedly, the abundance of Proteobacteria was significantly increased in RA patients (P< 0.05), and the abundance of Firmicutes, Fusobacteria and Verrucomicrobia were significantly reduced (P<0.05) at the level of Phylum (Figure 1). At the genus level, in the RA group, the abundance of Escherichia, Ruminococcus2 and Clostridium_sensu_stricto were significantly increased (P< 0.05), but the abundance of Lachnospiracea_incertae_sedis, Prevotella, Clostridium_XlVa, Roseburia, Dialister, Blautia, Megamonas and Gemmiger were significantly lower than the healthy controls (P< 0.05) (Figure 2). Moreover, Blautia, Anaerostipes and Ruminococcus2 have negative correlation with the absolute number of Tregs, and Cloacibacillus and Streptophyta have positive correlation with the absolute number of Th17.Conclusion:Patients with RA had a dysbiosis of the gut microbiota in both diversity and abundance, which is closely related to the impaired peripheral lymphocyte subsets, that may be related to the pathogenesis of RA, which might provide a new idea for RA treatment.References:[1]Wen HY, Wang J, Zhang SX, et al. Low-Dose Sirolimus Immunoregulation Therapy in Patients with Active Rheumatoid Arthritis: A 24-Week Follow-Up of the Randomized, Open-Label, Parallel-Controlled Trial.J Immunol Res2019;2019:7684352. doi: 10.1155/2019/7684352 [published Online First: 2019/11/30][2]Niu HQ, Li ZH, Zhao WP, et al. Sirolimus selectively increases circulating Treg cell numbers and restores the Th17/Treg balance in rheumatoid arthritis patients with low disease activity or in DAS28 remission who previously received conventional disease-modifying anti-rheumatic drugs.Clin Exp Rheumatol2019 [published Online First: 2019/05/11][3]Lee N, Kim WU. Microbiota in T-cell homeostasis and inflammatory diseases.Exp Mol Med2017;49(5):e340. doi: 10.1038/emm.2017.36 [published Online First: 2017/05/27]Acknowledgments:NoneDisclosure of Interests:None declared

scholarly journals AB0588 INFECTION AGGRAVATED DECREASE OF THE LEVEL OF TH17 AND TREG CELLS AND LOW-DOSE IL-2 REBALANCED TH17/TREG IN THE PERIPHERAL BLOOD OF PATIENTS WITH IDIOPATHIC INFLAMMATORY MYOPATHY

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1591.3-1591
Author(s):  
Y. Liang ◽  
H. Y. Wen ◽  
Y. Duan ◽  
Y. Liu ◽  
Z. Yu ◽  
...  
Keyword(s):  
T Cells ◽  
Low Dose ◽  
Lymphocyte Subsets ◽  
Absolute Number ◽  
Treg Cells ◽  
Idiopathic Inflammatory Myopathies ◽  
Inflammatory Myopathies ◽  
Infection Group ◽  
The Absolute ◽  
Organ Infection

Background:Idiopathic inflammatory myopathies (IIM) are featured by a series of clinical presentation such as proximal muscle weakness, increased serum levels of creatine kinase and other muscle enzymes and involvement of other organs and systems[1, 2], which results in high morbidity and early mortality[3]. We have known the changes of the level of Th17 and Treg cells in IIM in previous studies[4-6]. However, whether infection affects lymphocyte subsets or not and whether the effect of low-dose interleukin-2 (IL-2) can be influenced by the use of immunosuppressants or not are still unclear.Objectives:The study aimed to explore the changes of lymphocyte subsets in patients of IIM with or without important organ infection, and the restoration of Th17/Treg after receiving low-dose IL-2.Methods:A total of 118 IIM patients were enrolled and classified into infection group and non-infection group based on the important organ infection. Of them, 48 cases were treated with low dose IL-2 (5.0*105IU for 5 days). The absolute number of peripheral total T, B, CD4+T, CD8+T, NK, Th1, Th2, Th17 and Treg cell subsets were analyzed by flow cytometry combined with absolute counting beads. Clinical data, laboratory examinations and the levels of peripheral lymphocyte subsets were analyzed retrospectively.Results:In these patients, especially in the infection group, the absolute number of T, CD4+T, CD8+T, NK, Th1, Th2, Th17 and Treg cells were significantly decreased as compared with that in the healthy controls, which were significantly increased by low dose IL-2 (especially Treg cells) treatment. The levels of ESR, LDH and HBDH and the ratio of Th17/Treg were significantly lower than those before IL-2 treatment (Z=-2.237, -2.083, -2.140, -3.663,P=0.025, 0.037, 0.032, 0.000). The 48 cases who received IL-2 treatment were divided into 2 groups according to whether they used immunosuppressants. There was no significant difference in the absolute number of T, B, CD4+T, CD8+T, Th1, Th2, Th17 and Treg cells, the proportion of Th17 and Treg cells and the ratio of Th17/Treg between the 2 groups (P>0.05).Conclusion:Global decrease in lymphocyte subsets was found in IIM patients, especially those who had important organ infection. A significant re-balance of Th17/Treg was observed after receiving treatment with low-dose IL-2. Furthermore, the restoration of lymphocyte subsets showed similar degree after treatment with or without immunosuppressants. Low-dose IL-2 may become a potential therapy for IIM patients. The mechanism of lymphocyte decrease in IIM is required further to study.References:[1]Clark K E N, Isenberg D A. A review of inflammatory idiopathic myopathy focusing on polymyositis[J]. European Journal of Neurology, 2017.[2]Tieu J, Lundberg IE, Limaye V. Idiopathic inflammatory myositis. Best Pract Res Clin Rheumatol. 2016. 30(1): 149-68.[3]Mandel DE, Malemud CJ, Askari AD. Idiopathic Inflammatory Myopathies: A Review of the Classification and Impact of Pathogenesis. Int J Mol Sci. 2017. 18(5).[4]Zhang SX, Wang J, Sun HH, et al. Circulating regulatory T cells were absolutely decreased in dermatomyositis/polymyositis patients and restored by low-dose IL-2. Ann Rheum Dis. 2019 .[5]Espinosa-Ortega F, Gómez-Martin D, Santana-De Anda K, Romo-Tena J, Villaseñor-Ovies P, Alcocer-Varela J. Quantitative T cell subsets profile in peripheral blood from patients with idiopathic inflammatory myopathies: tilting the balance towards proinflammatory and pro-apoptotic subsets. Clin Exp Immunol. 2015. 179(3): 520-8.[6]Feng M, Guo H, Zhang C, et al. Absolute reduction of regulatory T cells and regulatory effect of short-term and low-dose IL-2 in polymyositis or dermatomyositis. Int Immunopharmacol. 2019. 77: 105912.Acknowledgments:Thanks for the support of my teachers, classmates and my family.Disclosure of Interests:None declared

scholarly journals Low expression of CD39 on regulatory T cells as a biomarker for resistance to methotrexate therapy in rheumatoid arthritis

2015 ◽  
Vol 112 (8) ◽  
pp. 2509-2514 ◽  
Author(s):  
Raphael Sanches Peres ◽  
Foo Y. Liew ◽  
Jhimmy Talbot ◽  
Vanessa Carregaro ◽  
Rene D. Oliveira ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
Regulatory T Cells ◽  
Joint Destruction ◽  
Healthy Controls ◽  
Suppressive Activity ◽  
First Line Therapy ◽  
Noninvasive Biomarker ◽  
A Prospective Study ◽  
Low Expression

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by joint destruction and severe morbidity. Methotrexate (MTX) is the standard first-line therapy of RA. However, about 40% of RA patients are unresponsive to MTX treatment. Regulatory T cells (Tregs, CD4+CD25+FoxP3+) are thought to play an important role in attenuating RA. To investigate the role of Tregs in MTX resistance, we recruited 122 RA patients (53 responsive, R-MTX; 69 unresponsive, UR-MTX) and 33 healthy controls. Three months after MTX treatment, R-MTX but not UR-MTX showed higher frequency of peripheral blood CD39+CD4+CD25+FoxP3+ Tregs than the healthy controls. Tregs produce adenosine (ADO) through ATP degradation by sequential actions of two cell surface ectonucleotidases: CD39 and CD73. Tregs from UR-MTX expressed a lower density of CD39, produced less ADO, and had reduced suppressive activity than Tregs from R-MTX. In a prospective study, before MTX treatment, UR-MTX expressed a lower density of CD39 on Tregs than those of R-MTX or control (P < 0.01). In a murine model of arthritis, CD39 blockade reversed the antiarthritic effects of MTX treatment. Our results demonstrate that MTX unresponsiveness in RA is associated with low expression of CD39 on Tregs and the decreased suppressive activity of these cells through reduced ADO production. Our findings thus provide hitherto unrecognized mechanism of immune regulation in RA and on mode of action of MTX. Furthermore, our data suggest that low expression of CD39 on Tregs could be a noninvasive biomarker for identifying MTX-resistant RA patients.

scholarly journals Reduction of peripheral regulatory T cells in active rheumatoid arthritis patients with coronary artery disease

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yanyan Wang ◽  
Rui Su ◽  
Baochen Li ◽  
Qiaoling Guo ◽  
Fangyuan Hu ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
T Cell ◽  
Active Rheumatoid Arthritis ◽  
Absolute Number ◽  
Treg Cells ◽  
T Cell Subsets ◽  
Cd4 T Cell ◽  
The Absolute ◽  
Artery Disease

Abstract Objective To identify lymphocyte and CD4 + T cell subset characteristics, particularly regulatory T cells (Tregs), in active rheumatoid arthritis (RA) patients with coronary artery disease (CAD). Methods A total of 54 RA patients with CAD (RA-CAD group), 43 RA patients without CAD (pure RA group), and 43 healthy controls (HC group) were enrolled. The absolute number and frequency of lymphocyte subpopulations and CD4 + T cell subsets were analyzed by flow cytometry. Serum levels of cytokines were analyzed using a cytometric bead array. Clinical and laboratory data were collected retrospectively and their correlation with CD4 + T subsets were analyzed. Results There was a significant decrease in the absolute number of Treg cells (CD4 + CD25 + Foxp3 + T cells) in the RA-CAD group compared to the pure RA group (p < 0.001). Similarly, both the absolute number (p = 0.001) and frequency (p = 0.011) of Tregs in the RA-CAD group were decreased compared to the HCs, causing a Th17/Treg imbalance (p = 0.044). No difference was found in the absolute number and frequency of Treg cells between the pure RA and HC groups. However, the absolute Th17 cell count was increased in the pure RA group (p = 0.032). The serum level of cytokine IL-17 was lower in the RA-CAD group than in the pure RA group (p = 0.023). In the RA-CAD group, the Treg number was negatively correlated with the RA disease activity score and ESR value, and LDL and ApoB100 levels were negatively correlated with the number of Th17 cells. Conclusions Active RA patients with CAD sustain more severe immune tolerance damage and Th17/Treg disorder. Monitoring of lymphocyte and CD4 + T cell subsets, particularly Treg cells, is crucial to understanding immune status in this group. Focusing on RA activity and CAD risk control, immune-regulatory therapy based on the Treg level may be more beneficial for RA patients with CAD.

scholarly journals POS0396 THE LEVEL OF PERIPHERAL REGULATORY T CELLS IS ASSOCIATED WITH THE CHANGES OF INTESTINAL MICROBIOTA IN PATIENTS WITH RHEUMATOID ARTHRITIS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 427-428
Author(s):  
R. Wu ◽  
J. An ◽  
T. Ding ◽  
H. Xue ◽  
X. F. Li ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
Intestinal Microbiota ◽  
Immune Tolerance ◽  
Cd4 T Cells ◽  
Absolute Number ◽  
Treg Cells ◽  
Genus Level ◽  
T Subsets ◽  
The Absolute

Background:Rheumatoid arthritis (RA) is a systemic autoimmunity inflammation disease characterized with chronic aggressive arthritis and the presence of abnormal antibodies. Several observations showed that the breakdown of immune tolerance caused by many complex interactions was involved in the development of RA[1]. However, the pathogenesis of RA remained unclear. It has been confirmed that the imbalance of Th17 and Treg cells play a crucial role in destroying immune tolerance [2]. Besides, researches showed that intestinal microbiota can influence host immunity by acting on the immune cells to play pro-inflammatory or anti-inflammatory effect, and in turn immune system can also regulate the microbiota[3, 4]. Thus, a frontier point of view in the field of rheumatism, immune microecology, was proposed, which is a novel concept for the breakdown of immune tolerance. Studies have confirmed that there was an imbalance of intestinal microbiota in patients with RA [4]. But the relationship between the CD4+T subsets cells and intestinal microbiota in RA is unknown.Objectives:We detected and compared the absolute number of CD4+T cells subsets in the peripheral blood and the proportion or abundance of intestinal microbiota in patients with RA and healthy adults, and then analyzed the relationship between them to explore the role of CD4+T cells subsets and intestinal microbiota in the pathogenesis of RA.Methods:We collected the sample of stool and blood from 15 patients with RA hospitalized at the Second Hospital of Shanxi Medical University and 8 age and gender-matched healthy controls(HC). The absolute number of CD4+T cells subsets including Th1, Th2, Th17 and Treg cells were detected by flow cytometry. The 16S rRNA in the stool specimens were sequenced by the Roche/45 high-throughput sequencing platform. We analyzed whether there was correlarion between CD4+T subsets cells and intestinal microbiota.Results:Patients with RA had a higher level of Christensenellaceae and a lower level of Pseudomonadaceae as compared with those of HCs at the family level (p<0.05). And at the genus level, the patients with RA had higher levels of Ruminococcus torques, Christensenellaceae R-7, Ruminiclostridium 9 and Ruminococcus 1 compared with those of HCs (p<0.05) (Figure 1).And the Ruminococcus torques at the genus level was negative correlated with the absolute number of Treg cells (p<0.001) (Figure 2).Conclusion:The results here suggested that there were different proportion or abundance of intestinal microbiota between the patients with RA andHCs. And the changes of intestinal microbiota such as Ruminococcus torques were associated with Treg cells, further indicating that the imbalance of intestinal microbiota in RA can destory the immune tolerance. The above results uncovered that the intestinal microbiota had immunomodulatory function, which may be the upstream mechanism participated in the pathogenesis of RA.References:[1]Weyand CM, Goronzy JJ. The immunology of rheumatoid arthritis. Nat Immunol 2021, 22(1): 10-18.[2]Weyand CM, Goronzy JJ. Immunometabolism in the development of rheumatoid arthritis. Immunol Rev 2020, 294(1): 177-187.[3]Brown EM, Kenny DJ, Xavier RJ. Gut Microbiota Regulation of T Cells During Inflammation and Autoimmunity. Annu Rev Immunol 2019, 37: 599-624.[4]du Teil Espina M, Gabarrini G, Harmsen HJM, Westra J, van Winkelhoff AJ, van Dijl JM. Talk to your gut: the oral-gut microbiome axis and its immunomodulatory role in the etiology of rheumatoid arthritis. FEMS Microbiol Rev 2019, 43(1).Figure 1.At the family level (a-b) and the genus level(c-f), the relative abundance of intestinal microbiota in patients with RA and HCs were different. Data were expressed as median (Q1, Q3) and analyzed by Wilcoxon test. (*** P < 0.001, **P < 0.01 and *P < 0.05).Figure 2.A heatmap shows the correlation between the intestinal microbiota and CD4+T cells in patients with RA, and Ruminococcus torques at the genus level was negative related with Treg cells. (Colors indicate the Spearman rank correlation, *** P < 0.001).Disclosure of Interests:None declared

Fecal Microbiome and Bile Acid Metabolome in Adult Short Bowel Syndrome

2021 ◽  
Author(s):  
Harold J. Boutte ◽  
Jacqueline Chen ◽  
Todd N. Wylie ◽  
Kristine M. Wylie ◽  
Yan Xie ◽  
...  
Keyword(s):  
Bile Acid ◽  
Gut Microbiota ◽  
Short Bowel Syndrome ◽  
Lithocholic Acid ◽  
Intestinal Failure ◽  
Patient Specific ◽  
Rrna Gene ◽  
Healthy Controls ◽  
Fecal Microbiome ◽  
Short Bowel

Background & Aims: Loss of functional small bowel surface area causes short bowel syndrome (SBS), intestinal failure, and parenteral nutrition (PN) dependence. The gut adaptive response following resection may be difficult to predict, and it may take up to two years to determine which patients will wean from PN. Here we examined features of gut microbiota and bile acid (BA) metabolism in determining adaptation and ability to wean from PN. Methods: Stool and sera were collected from healthy controls and from SBS patients (n=52) with ileostomy, jejunostomy, ileocolonic and jejunocolonic anastomoses fed with PN plus enteral nutrition or who were exclusively enterally fed. We undertook 16S rRNA gene sequencing, BA profiling and 7α-hydroxy-4-cholesten-3-one (C4) quantitation with LC-MS/MS, and serum amino acid analyses. Results: SBS patients exhibited altered gut microbiota with reduced gut microbial diversity compared to healthy controls. We observed differences in the microbiomes of SBS patients with ileostomy vs. jejunostomy, jejunocolonic vs. ileocolonic anastomoses, and PN-dependence compared to those who weaned from PN. Stool and serum BA composition and C4 concentrations were also altered in SBS patients, reflecting adaptive changes in enterohepatic BA cycling. Stools from patients who weaned from PN were enriched in secondary BAs including deoxycholic acid and lithocholic acid. Conclusions: Shifts in gut microbiota and BA metabolites may generate a favorable luminal environment in select SBS patients, promoting the ability to wean from PN. Pro-adaptive microbial species and select BA may provide novel targets for patient-specific therapies for SBS.

scholarly journals IgA-deficient humans exhibit gut microbiota dysbiosis despite secretion of compensatory IgM

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jason R. Catanzaro ◽  
Juliet D. Strauss ◽  
Agata Bielecka ◽  
Anthony F. Porto ◽  
Francis M. Lobo ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
16S Rrna Gene Sequencing ◽  
Iga Deficiency ◽  
Secretory Iga ◽  
Rrna Gene ◽  
Healthy Controls ◽  
Microbiota Composition ◽  
Antibody Isotype ◽  
Selective Iga Deficiency ◽  
Gut Microbiota Composition

Abstract Immunoglobulin A is the dominant antibody isotype found in mucosal secretions and enforces host-microbiota symbiosis in mice, yet selective IgA-deficiency (sIgAd) in humans is often described as asymptomatic. Here, we determined the effects of IgA deficiency on human gut microbiota composition and evaluated the possibility that mucosal secretion of IgM can compensate for a lack of secretory IgA. We used 16S rRNA gene sequencing and bacterial cell sorting to evaluate gut microbiota composition and taxa-specific antibody coating of the gut microbiota in 15 sIgAd subjects and matched controls. Despite the secretion of compensatory IgM into the gut lumen, sIgAd subjects displayed an altered gut microbiota composition as compared to healthy controls. These alterations were characterized by a trend towards decreased overall microbial diversity as well as significant shifts in the relative abundances of specific microbial taxa. While secretory IgA in healthy controls targeted a defined subset of the microbiota via high-level coating, compensatory IgM in sIgAd subjects showed less specificity than IgA and bound a broader subset of the microbiota. We conclude that IgA plays a critical and non-redundant role in controlling gut microbiota composition in humans and that secretory IgA has evolved to maintain a diverse and stable gut microbial community.

scholarly journals Comparative Analysis of Fecal Microbiota Composition Between Rheumatoid Arthritis and Osteoarthritis Patients

Genes ◽  
2019 ◽  
Vol 10 (10) ◽  
pp. 748 ◽  
Author(s):  
Jin-Young Lee ◽  
Mohamed Mannaa ◽  
Yunkyung Kim ◽  
Jehun Kim ◽  
Geun-Tae Kim ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Gut Microbiota ◽  
Gut Microbiome ◽  
Bacterial Species ◽  
Amplicon Sequencing ◽  
Fecal Microbiota ◽  
Rrna Gene ◽  
Microbiota Composition ◽  
Stool Samples ◽  
Gut Microbiota Composition

The aim of this study was to investigate differences between the gut microbiota composition in patients with rheumatoid arthritis (RA) and those with osteoarthritis (OA). Stool samples from nine RA patients and nine OA patients were collected, and DNA was extracted. The gut microbiome was assessed using 16S rRNA gene amplicon sequencing. The structures and differences in the gut microbiome between RA and OA were analyzed. The analysis of diversity revealed no differences in the complexity of samples. The RA group had a lower Bacteroidetes: Firmicutes ratio than did the OA group. Lactobacilli and Prevotella, particularly Prevotella copri, were more abundant in the RA than in the OA group, although these differences were not statistically significant. The relative abundance of Bacteroides and Bifidobacterium was lower in the RA group. At the species level, the abundance of certain bacterial species was significantly lower in the RA group, such as Fusicatenibacter saccharivorans, Dialister invisus, Clostridium leptum, Ruthenibacterium lactatiformans, Anaerotruncus colihominis, Bacteroides faecichinchillae, Harryflintia acetispora, Bacteroides acidifaciens, and Christensenella minuta. The microbial properties of the gut differed between RA and OA patients, and the RA dysbiosis revealed results similar to those of other autoimmune diseases, suggesting that a specific gut microbiota pattern is related to autoimmunity.

Enrichment of Mononuclear Cells From Cryopreserved Cord Blood Units Using the Purecell” Select System.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2159-2159
Author(s):  
Bryan P Spencer ◽  
Indreshpal Kaur ◽  
Patrick Fong ◽  
Safa Karandish ◽  
Nirmali Ponweera ◽  
...  
Keyword(s):  
T Cells ◽  
Cord Blood ◽  
Mononuclear Cells ◽  
Cellular Therapy ◽  
Standard Procedure ◽  
Absolute Number ◽  
Common Procedure ◽  
Expansion Time ◽  
Significant Difference ◽  
The Absolute

Abstract Abstract 2159 Poster Board II-136 INTRODUCTION: Many procedures for manufacturing clinical products for cellular therapy involve enrichment of mononuclear cells (MNC). The most common procedure is density gradient separation. Disadvantages of this procedure are low yield of cells especially with cryopreserved products and the open events during processing. We evaluated use of the Purecell” Select System (PALL Medical, NY) for enriching MNC from cryopreserved Cord Blood Units (CBU). METHODS: Initial experiments were performed to optimize the system for recoveries of total nucleated cells (TNC), MNC, neutrophils, lymphocytes, monocytes and CD34+/CD3+ cells. We evaluated thaw/predilute/filter vs thaw/filter, starting volumes (30 — 95mls) and three different methods for harvesting cells from the filter (standard method, input bag rinse and harvest port rinse). Once conditions were optimized, cryopreserved CBU were thawed and split into two fractions. One half of the product was diluted and processed on the Purecell” Select System. The other half was washed and ficolled. The MNC fraction was CD3+ enriched using CD3/28 beads and then cultured with rIL-2 (200 units/ml) for 14 days. The absolute number of CD3+ cells post culture, fold expansion and viabilities of these cells were determined. RESULTS: The Purecell” procedure was 15 times faster than the ficoll method. Optimal volume to load onto the filter was 50ml. When MNC were harvested by the three recommended procedures, there was no difference in recoveries of TNC, MNC, CD34+ and CD3+ cells and neutrophils. However, the lymphocyte and monocyte recoveries were higher (p<0.05 and p=0.001) when harvested with Input bag rinse compared to the standard procedure. Monocyte recoveries were also higher with the harvest port rinse (p=0.004) when compared to the standard procedure. The direct comparison studies of the two MNC enrichment systems demonstrated that the Purecell” Select System gave significantly higher recovery of TNC (p= 0.003), MNC (p=0.029), CD34+ cells (p<0.001) and granulocytes (p<0.001). There was no statistical difference in T cell recoveries, however, there was a significant difference in the recovery of T cells after CD3/28 enrichment.. Interestingly, T cells began to proliferate earlier from the PALL system compared to ficoll isolated T cells (day 4 vs day 7). Although the fold expansion was greater for the ficolled prepared cells, the absolute numbers of T cells obtained after 14 days of culture with rIL-2 was greater for the Purecell Select System in all experiments. The viabilities of the cells from both cultures were comparable. CONCLUSIONS: PALL's Purecell” Select System can be used for clinical processing since it is a functionally closed system. The advantage of this system compared to the ficoll method are the reduced time for processing, increase yield in T cells (post processing), the earlier expansion time These benefits result in an increase in absolute number of T cells in post culture. A clinical trial using this system is about to be initiated. Disclosures: Karandish: Pall Medical: Employment. McMannis:Pall Medical: Research Funding.

Real-Time Immunophenotyping of Peripheral Blood Early Post Myeloablative Cord Blood Transplant Can Identify Patients at Risk for Primary Graft Failure

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3897-3897
Author(s):  
Jianqiang Li ◽  
Filippo Milano ◽  
Joseph M Blake ◽  
David C. Oliver ◽  
Ian Nicoud ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Graft Failure ◽  
De Novo ◽  
Nk Cell ◽  
Cell Subset ◽  
Absolute Number ◽  
Hematopoietic Recovery ◽  
Primary Graft Failure ◽  
The Absolute

Abstract Introduction: Primary graft failure (PGF) is a potentially life threatening complication following hematopoietic cell transplantation. Patients undergoing cord blood transplantation (CBT) are at higher risk for PGF and also experience delayed hematopoietic recovery. The ability to distinguish between PGF and delayed engraftment is critical for correct clinical management. Limited data exists analyzing the kinetics of engraftment of specific circulating cell lineages within 14 days after double CBT (dCBT). Herein, we investigate the potential for real time immunophenotyping (RTIP) of day 7 and day 14 post-transplant peripheral blood (PB) samples as an effective and economically feasible tool to distinguish PGF versus delayed engraftment. Methods: Between Feb 2013 and Jun 2014, 26 patients underwent a myeloablative dCBT at our institute. Heparinized PB samples (30 ml) were obtained from patients on days 7 and 14 post-transplant. PB mononuclear cells (PBMCs) were isolated by density gradient separation and total cell number was counted. RTIP with 9-color flow cytometry was performed at each time point using isolated fresh PBMC. Results: Median time to engraftment was 19 days (range 12-51) in 23 evaluable patients. The remaining 3 patients had no documented hematopoietic recovery by routine daily CBCs. Two patients were declared PGFs when day 21 PB chimerism results demonstrated 100% host T cells followed by confirmation of 0% donor engraftment on day 28. The third patient never had sufficient quantity of circulating cells to obtain RTIP results and died on day 28 with no hematopoietic recovery. Excluding this patient, we were able to quantify the absolute number of PBMC at day 7 (median: 3.1/µl; 95% CI: 2.6-4.6) and day 14 (median: 26/µl; 95% CI: 33-103) in all other patients. There was an increase in the absolute number of PBMC from day 7 to 14 in all patients except the two who experienced PGF. Furthermore, RTIP of day 7 PBMC revealed a predominance of T cells that were donor-derived, while day 14 RTIP of PBMC demonstrated a decreased frequency of T cells and increased frequency of predominantly donor-derived monocytes and NK cells. In contrast to the engrafting patients, the two PGF patients displayed a markedly different pattern in RTIP with minimal evidence of circulating monocytes in the day 14 samples (Fig 1A). Importantly, RTIP demonstrated that day 7 PBMC contained a higher frequency of CD14+CD16- monocytes and CD56brightCD16- NK cells than were infused, suggesting these specific cells were generated de novo and were not representative of cells infused with the graft (data not shown). Correlations between day 7 or day 14 cell subset numbers and time to engraftment were analyzed. The median absolute number of monocytes at day 7 was 0.075/µl (95% CI: 0.03-0.153). Patients with day 7 monocyte counts above the median demonstrated earlier engraftment than patients below the median (17.5 vs 26.5 days; p= 0.011). Using linear regression with engraftment as the variable of interest and the absolute number of day 7 monocytes as the predictor, the coefficient was 0.6 (95% CI: 0.075- 0.78, P=0.025) (Fig 1B). As expected, higher day 14 monocytes also correlated with earlier engraftment. With respect to the NK cell subset, the absolute number of NK cells at day 7 was not significantly correlated with engraftment time, but day 14 NK cell numbers were predictive of engraftment kinetics. The median number of NK cells at day 14 was 2.78/µl (95% CI: 1.87-5.79). Patients with day 14 NK counts above the median had earlier engraftment than those below the median (16 vs 26.5 days; (P=0.0034). The regression coefficient was 0.5 (95% CI, 0.077- 0.77; P=0.024). Finally, although total T cell numbers at day 7 and day 14 had no correlation with engraftment time, the two evaluable PGF patients had a greater inversion of CD4:CD8 ratio (0.038 & 0.058) than others (median: 0.71, 95% CI: 0.22-1.42) at day 14. Conclusions: This study provides the first clear evidence that RTIP of PBMC at days 7 and 14 can detect the kinetics of circulating de novo generated monocytes and NK cells and also reflects the in vivo immune environment in patients following dCBT. Importantly, detecting and measuring specific cell subsets using RTIP permits earlier identification of patients at high risk of graft failure versus patients with delayed engraftment and warrants further development as a clinically feasible diagnostic method to guide clinical intervention. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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