For instance, we found that the memory CD25NEG, but not the memor

For instance, we found that the memory CD25NEG, but not the memory CD25INT cells, were associated with chronic immune responses and were expanded on SLE patients (Fig. 2 and 3). This suggests that the CD25NEG memory population may play a role in auto-immune disease. In summary, we report in this study that a large percentage of memory CD4+ T cells in humans express intermediate levels of CD25. CD25 expression on the CD25INT memory population appears to be important biologically and that the CD25INT population is greatly affected by IL-2 immunotherapy in cancer patients. These findings not only improve our understanding of

the role of CD25 in human immunology, but may also have clinical implications by helping to illuminate the mechanisms and potentially improve the efficacy of therapies that target IL-2 and CD25. Human PBMCs were isolated by centrifugation of heparinized blood over Ficoll-Plaque™ PLUS (GE Healthcare). Isolated PBMCs were either analyzed fresh or were frozen in 45% RPMI/45%

FBS/10% DMSO and then thawed for analysis. CT99021 purchase Staining for flow cytometry was done at either 4°C or room temperature for 30 min with: CD3 (UCHT1), CD4 (SK3), CD8 (SK2), CD25 (Miltenyi, 4E3), CD25 (BD, M-A251), CD95 (DX2), CD45RA (HI100), CD45RO (UCHL1), CD127 (eBioRDR5), CD28 (CD28.2), CD134 (ACT35), CCR5 (2D7/CCR5), or CD319 (162.1). For intracellular staining, cells prepared with Foxp3 Staining Buffer Set (eBioscience) according to the manufacturer’s instructions

and incubated at either 4°C or room temperature for 30 min with: EOMES allophycocyanin (WD1928), FOXP3 (236A/E7), Ki67 (B56), pSTAT5 (47), IL-17A (BL168), Granzyme B (GB11), BCL-2 (100), IL-2 (MQ1-17H12), or IFN-γ (B27). Antibodies were acquired from Miltenyi, eBioscience, BD Biosciences, BioLegend, Invitrogen, and Beckman Coulter. All samples were run on an LSR II flow cytometer or FACSAria II and analyzed by FlowJo or Winlist. Sorting experiments were done using CD4+ cells enriched by Miltenyi LS columns from fresh PBMCs that were stained and sorted using a BD FACSAria II Cell Sorter. PBMCs from Thymidylate synthase individuals (ten females, five males; mean age, 36; age range, 27–61) without known autoimmune disease or cancer were used as healthy donors in this study. Patients with SLE (ten females; mean age, 40; age range, 20–49) that took part in the study fulfilled the American College of Rheumatology revised classification criteria for lupus [54]. Patients had active (n = 7) or inactive (n = 3) renal nephritis and were being treated with a variety of drugs (hydroxychloroquine n = 9, mycophenolate n = 4, prednisone n = 7).

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