Purity Of CD4 Research Articles

Characterization of an in vitro model to study CD4+ T cells metabolism in dairy cows

Dairy cows are susceptible to several health disorders throughout their lactation. Objectives were to characterize an in vitro model to study bioenergetic measures in CD4+ T lymphocytes in dairy cows. Twenty-four healthy mid-lactating multiparous Holstein dairy cows were enrolled at a mean (±standard deviation) of 234 ± 22 d in milk (DIM). Cows were blocked according to DIM and blood was collected to isolate peripheral blood mononuclear cells followed by magnetic separation of CD4+ T lymphocytes using bovine specific monoclonal antibodies. The isolated CD4+ T lymphocytes from each cow were split into 2 tubes and randomly assigned to incubate in an assay medium as control (CON) or with a combination of phorbol myristate acetate and ionomycin (PMA+IMY) to evaluate metabolic function under resting and activated state. Mitochondrial and glycolytic functional kinetics were recorded in CD4+ T lymphocytes based on real-time measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) under basal conditions and in response to complex V inhibitor (oligomycin), a protonophore uncoupler (BAM 15), and complex I and complex III inhibitors (rotenone and antimycin A). The mean (±SD) viability and purity of CD4+ T lymphocytes was 92.5 ± 2.9% and 95.2 ± 2.9%, respectively. The basal OCR in CD4+ T lymphocytes treated with PMA+IMY was greater than CON; nevertheless, the maximal respiration rate (CON = 58.0 vs. PMA+IMY = 47.3 ± 5.7 pmol/min) and sparing respiratory capacity rate (CON = 42.0 vs. PMA+IMY = 28.7 ± 4.2 pmol/min) were decreased in activated CD4+ T lymphocytes. The ECAR in CD4+ T lymphocytes increased progressively over time in PMA+IMY compared with CON, which indicated an increase in aerobic glycolysis in PMA+IMY compared with CON (CON = 46.9 vs. PMA+IMY = 86.4 ± 7.0 pmol/min). Activated CD4+ T lymphocytes exhibit a metabolic switch from oxidative phosphorylation to aerobic glycolysis, which may support rapid cell proliferation. The results observed in this experiment demonstrate the sensitivity of the technique to detect changes in metabolic function under different cellular conditions, providing a robust framework to study immuno-metabolism in dairy cattle.

Ectopic Expression of TIGIT in Lung Adenocarcinoma and Its Clinical Significance.

This study aimed to investigate the T cell immunoreceptor with ITIM and Ig domains (TIGIT) expression in lung adenocarcinoma (LUAD). TIGIT expression was measured by western blot, reverse transcription quantitative polymerase chain reaction. Seventy-two paired surgical specimens were collected from patients with stage I-IV LUAD. The expression of TIGIT in surgical specimens was determined using immunohistochemistry. TIGIT was overexpressed in LUAD tissues. Moreover, overexpressed TIGIT was significantly associated with advanced clinical staging, lymph node metastasis, distant metastasis, and TP53 mutations in LUAD. Moreover, high expression of TIGIT was negatively correlated with purity of CD4+ T cells. High rations of TIGIT+CD4+ T cells predicted poor overall survival of LUAD patients. Additionally, high ratios of TIGIT+CD4+ T cells is closely related to CD4+ T cell depletion. Taken together, TIGIT was overexpressed in LUAD patients. High levels of TIGIT induced the alteration of CD4+ T cell based immunomodulation and predicted poor prognosis of LUAD patients. Therefore, TIGIT can be potential biomarker for LUAD.

Methyl-cpg-binding Domain Protein 2 Silencing Inhibits Th17 Differentiation of CD4+T cells Induced by Ovalbumin.

Little is known about MBD2's epigenetic regulation in the immune pathogenesis of CD4+T cell differentiation. This study attempted to explore the mechanism of methyl-cpg-binding domain protein 2 (MBD2) in CD4+T cell differentiation stimulated by environmental allergen ovalbumin (OVA). Mononuclear cells were separated from the spleen tissues of male C57BL/6 mice. The OVA interfered with the differentiation of splenic mononuclear cells and CD4+T cells. The CD4+T cells were obtained by magnetic beads and identified by CD4 labeled antibody. CD4+T cells were transfected with lentivirus to silence MBD2 gene. A methylation quantification kit was used to detect 5-mC levels. The purity of CD4+T cells reached 95.99% after magnetic beads sorting. Treatment with 200 μg/mL OVA stimulated the CD4+T cells differentiation to Th17 cells and promoted the secretion of IL-17. After being induced, the Th17 cell ratio increased. 5-Aza inhibited the Th17 cell differentiation and the IL-17 level in a dose-dependent manner. Under the intervention of the Th17 induction and 5-Aza, MBD2 silencing inhibited the differentiation of Th17 cell, and decreased the IL-17 and 5-mC levels in the cell supernatants. MBD2 silencing reduced the scale of the Th17 cell and IL-17 levels in the OVA-treated CD4+T cells. MBD2 affected IL-17 and 5-mC levels by mediating the Th17 cell differentiation in splenic CD4+T cells that were interfered with 5-Aza. OVA induced Th17 differentiation and increased IL-17 levels, inhibited by MBD2 silencing.

A closed-culture system using a GMP-grade culture bag and anti-CD3/28 coated bead stimulation for CD4+ T cell expansion from healthy and HIV-infected donors

CD4 immunotherapy is potentially useful in immune reconstitution of CD4+ T cells for HIV-infected patients. Transfusion of anti-CD3/28 expanded CD4+ T cells is also proved to be safe and effective in both SIV-infected macaques and HIV-infected patients. However, there is no such standardized and practical protocol available for cell production in order to use in clinics. This study thus aimed to develop a closed-culture system for in vitro CD4+ T lymphocyte expansion by using a commercially available GMP-grade culture bag and anti-CD3/28 activation. Freshly isolated CD4+ T cells by immunorosette formation from healthy donors and cryopreserved CD4+ T cells from HIV-infected patients with CD4 count over 500 cells/μL were stimulated with anti-CD3/28 coated beads. The activated cells were then expanded in conventional culture flasks and GMP-grade culture bags for three weeks. Fold expansion, cell viability, growth kinetic and phenotypic characters were observed. Results revealed that purified CD4+ T cells from healthy individuals cultured in flasks showed better expansion than those cultured in bags (797-fold and 331-fold, respectively), whereas, their cell viability, growth kinetic and expanded CD4+ T cell purity were almost similar. A large-scale production was also conducted and supported consistency of cell proliferation in the closed-culture system. Frozen CD4+ T lymphocytes from the patients were able to remain their growth function and well expanded with a good yield of 415-fold, 85% viability and 96% purity of CD4+ T cells at the end of a 3-week culture in bags. This developed closed-culture system using culture bags and anti-CD3/28 coated beads, therefore, can achieve a large number of expanded CD4+ T lymphocytes with good reproducibility, suggesting a promising protocol required for adoptive immunotherapy.

Effect evaluation on sorting method of CD4+T lymphocytes

Objective To establish an accurate sorting method with less interference to the cell activity for isolating CD4+T lymphocytes.Methods Human peripheral blood cells were sorted by density gradient centrifugation (DGC) and magnetic activated cell sorting (MACS) in turn,and then the cell purity,morphological observation and survival rate of CD4+T lymphocytes were adopted to conduct the evaluation on the sorting method.Results The purity of CD4+T lymphocytes was (38.8 ± 2.7)％,while the purity of CD4+T lymphocytes was (96.2 ± 0.7)％ after magnetic activated cells being sorted,the difference between them was statistically significant (P ＜ 0.01).The activity was the highest within 24 hours with intact cell shape and function.Conclusion The Percoll DGC combined with the MACS can collect highly pure CD4+T lymphocytes with less influence on the cell activity and shape,the sorted cells can continue to be used for related researches. Key words: CD4+T lymphocytes; Cell sorting; Magnetic-activated cell sorting

Isolation, amplification and identification of human natural CD4⁺CD25⁺ regulatory T cells in vitro

To establish a method for in vitro expansion of human natural CD4⁺CD25⁺ T regulatory cell (Treg) cells for clinical study and immunotherapy. Human natural CD4⁺CD25⁺ Treg were isolated from peripheral blood monocyte cells (PBMCs) by magnetic activated cell sorting (MACS) and expanded by CD3/CD28 expansion beads, IL-2 and rapamycin. The number and the viability of the freshly isolated and expanded Treg were detemined by trypan blue staining. The phenotype and the purity of the freshly isolated and expanded Treg were analyzed by FACS. Treg suppression activity was assessed by mixed lymphocyte reaction (MLR) assay. Human natural Treg were expanded up to 2 000 folds after 3 weeks in culture, and the activity was more than 97%. The expanded Treg retained Treg phenotype as shown by their freshly isolated counterparts, and the purity of CD4⁺CD25⁺FoxP3⁺ Treg was (94.22 ± 2.12)%. The expanded Treg demonstrated a similar potent suppression of both proliferating auto- and allo- CD4⁺CD25⁻ effector T cells in vitro in a cell number-dependent manner. An in vitro expansion of human natural Treg was established to obtain large numbers of human Treg with highly suppressive phenotype and function, thereby providing a solution to the availability of sufficient human natural Treg in clinical study and immunotherapy.

Photodegradable hydrogels for capture, detection, and release of live cells.

Cells may be captured and released using a photodegradable hydrogel (photogel) functionalized with antibodies. Photogel substrates were used to first isolate human CD4 or CD8 T-cells from a heterogeneous cell suspension and then to release desired cells or groups of cells by UV-induced photodegradation. Flow cytometry analysis of the retrieved cells revealed approximately 95% purity of CD4 and CD8 T-cells, suggesting that this substrate had excellent specificity. To demonstrate the possibility of sorting cells according to their function, photogel substrates that were functionalized with anti-CD4 and anti-TNF-α antibodies were prepared. Single cells captured and stimulated on such substrates were identified by the fluorescence "halo" after immunofluorescent staining and could be retrieved by site-specific exposure to UV light through a microscope objective. Overall, it was demonstrated that functional photodegradable hydrogels enable the capture, analysis, and sorting of live cells.

Photodegradable Hydrogels for Capture, Detection, and Release of Live Cells

AbstractCells may be captured and released using a photodegradable hydrogel (photogel) functionalized with antibodies. Photogel substrates were used to first isolate human CD4 or CD8 T‐cells from a heterogeneous cell suspension and then to release desired cells or groups of cells by UV‐induced photodegradation. Flow cytometry analysis of the retrieved cells revealed approximately 95 % purity of CD4 and CD8 T‐cells, suggesting that this substrate had excellent specificity. To demonstrate the possibility of sorting cells according to their function, photogel substrates that were functionalized with anti‐CD4 and anti‐TNF‐α antibodies were prepared. Single cells captured and stimulated on such substrates were identified by the fluorescence “halo” after immunofluorescent staining and could be retrieved by site‐specific exposure to UV light through a microscope objective. Overall, it was demonstrated that functional photodegradable hydrogels enable the capture, analysis, and sorting of live cells.

Whole Exome Sequencing and Analysis Of Mutations In Sézary Syndrome

Introduction and RationaleSézary Syndrome (SS), the leukemic variant of cutaneous T-cell lymphoma CTCL), is characterized by generalized erythroderma and >1000 circulating central memory T-cells with a CD4+CD26- phenotype, Other clinical features include reactive adenopathy, keratoderma, ectropion, pruritus, and colonization with Staphylococcus aureus. SS can arise de novo or emerge in patients with Mycosis Fungoides (MF). Morphologically, SS cells were first recognized by their convoluted nuclei and karyotype analysis has shown the presence of multiple chromosomal abnormalities and copy number variants, none of which are disease defining; however, the mutational landscape of SS has not been investigated systematically. Here we present initial findings from profiling somatic mutation by whole exome sequencing. MethodsIn order to delineate the genomic mutation profile of Sézary cells we compared protein coding sequences from CD4+ and CD4- peripheral blood cell DNA to that from autologous cultured fibroblasts derived from ten newly diagnosed SS patients with high percentages of circulating SS cells. CD4+ T-cells were selected by a Miltenyi Biotec CD4+ T Cell Isolation Kit and genomic DNA was extracted from CD4+ T-cells, residual CD4- cells, and autologous fibroblasts from each patient using an All Prep DNA/RNA/Protein Mini Kit (Qiagen). Purity of CD4+ T-cell sorting confirmed by flow cytometry was > 90% purity. Skin biopsies from SS non-lesional skin were explanted, cultured for 5-6 weeks, and used to extract genomic control DNA for each patient. Whole exome DNA was enriched by capture using a Nimblegen VCRome version 2.1 array. Nine billion bases of sequence was generated for each sample on an Illumina HiSeq 2000 run for 101 cycles. This resulted in approximately 95% target covered to a minimum depth of 20 bases, with and overall average coverage of over 130 bases in all samples. Somatic mutations were called by comparing sequence from all tissues to the human reference sequence (GRC37) and then subtracting nucleotide variation found in the fibroblast lines from variation found in the CD4- and CD4+ cell populations. ResultsWe found an average of 82 mutations per patient in the CD4+ cells. TP53 with 4 mutations was the most commonly mutated, three patients harbored MSH3 mutations, which may contribute to higher mutation frequencies in those patients. Singleton mutations in MYC, MLLT4 are among cancer-related transcription factors, and singleton NF1 and Notch4 suggest aberrant signaling. Surprisingly, the CD4- cells also harbored TP53 mutations in 3 of the 4 patients. ConclusionsWhole exome sequencing revealed recurrent mutations in two genes: TP53 and MSH3 and hinted at involvement of RAS and Notch pathway signaling, although no RAS mutations were observed. The presence of TP53 mutation in CD4- cells was unanticipated, and may result from mutation of a common progenitor cell population in the CD4- cells. Studies are underway, including analysis of copy number gains and losses to further elucidate the mutational patterns in SS patients. Disclosures:No relevant conflicts of interest to declare.

Purification and Expansion of Antigen-Specific Human Tregs for Clinical Application Using a Single Step CD25 Positive Selection

AbstractAbstract 1899 Background:Regulatory T cells (Tregs) play a role in maintaining T cell homeostasis and self-tolerance. Tregs immunotherapy has great potential for the prevention of allograft rejection and graft-vs-host disease (GVHD), but GMP reagents and procedures are required for clinical use. We have shown that allo-specific Tregs can be isolated by positive CD25 selection and negative selection of CD127, CD8 and CD19 by Miltenyi immunobeads, and then expanded in amounts sufficient for potential clinical use. Such a number of immunobeads would make a GMP procedure so costly to be clinically unaffordable. Therefore, in this study we tested whether a single step of CD25 positive selection using magnetic micro beads followed by expansion with allogeneic DC, rapamycin, IL-2, and IL-15 for 12 days generates Tregs that retain their phenotype (Foxp3) expression to a high level, are minimally contaminated with non-Tregs, and are highly suppressive.We explored a simplified approach that employs only CD25 positive selection, followed by culture. Concerns still remain on the purity and contamination of the expanded population, in particular contamination with cytotoxic CD8+T cells as these cells could have unfavorable outcomes in immunotherapy trials. Methods:We purified Tregs using the ‘clinical grade' CD25 human positive selection magnetic beads from Miltenyi; we then labeled the Tregs with CFSE and cultured with allogeneic monocyte-derived DC as stimulus in the presence of rapamycin, IL-2 and IL-15. At the end of 12 days expanded allo-specific Tregs were analyzed for their phenotype, sorted and tested for alloreactive and suppressive functions. We performed a total of 10 experiments: Results:The purity of CD4+CD25+CD127dim Foxp3+after the initial separation ranged from 20–83%, median-44%.At the end of expansionRangedMedianCD25+Foxp3+46–82%75%CD8+0–2%0.8%B cells0–11%1%NK cells1–12%2%Monocytes1–6%2%Myeloid DC0–7%1%To assess whether the non-Tregs in the culture were allo-reactive we sorted the expanded Tregs into: CFSE+CD25+, CFSE−CD25−, CFSE+CD25−, CFSE−CD25+, we then set up a 3 and 6 days mixed leukocyte reaction (MLR) against the initial stimulator or self. Non-Tregs in the culture did not respond to antigen with or without IL-2, whereas the allo-reactive Tregs, CFSE−CD25+, responded to allogeneic DC only in the presence of IL-2. CFSE+ Tregs did not respond even in the presence of IL-2. To determine whether the entire expanded population was enriched with antigen specific suppression, we culture the enriched ‘Tregs' at various ratios with naïve autologous CD4+CD25−responder T cells and allogeneic DC from original stimulator, and found that a 50% suppression was achieved with a 1:2000 Treg:Tconv ratio. To analyze fidelity of cytokine secretion profile, we re-stimulated cultured ‘Tregs' with allogeneic DC or PMA/Iononycin and compared data to expanded Tconv from the same individual; Tregs had significantly more TGF-beta and less TNF-alpha secretion than Tconv. Conclusion:Our data demonstrate that Tregs purified with the GMP-compatible CD25 Miltenyi immunobeads can be expanded, retain high levels of Foxp3 expression, and maintain high suppressive potency. The frequency of contaminating cells after culture was small and we did not observed residual alloreactivity. Disclosures:No relevant conflicts of interest to declare.

MICRORNA EXPRESSION AND IDENTIFICATION OF CD4+T LYMPHOCYTES IN PATIENTS WITH ACUTE CORONARY SYSDROME

ObjectivesTo screen differential microRNA expression profiles of CD4+T lymphocyte from the patients with acute coronary syndrome (ACS) and the healthy controls by microarray analysis technique. To elucidate the mechanism responsible...

Screening and function identification of CD4 + CD25 - T cells induced by immature dendritic cells transfected with IKK2dn

Objective To investigate the method of screening CD4 + CD25 - T cells from regulatory T cells (Treg) induced by recipient-derived immature dendritic cells (imDC) transfected by IKK2dn and loaded with donor antigens,and assess their immunologic function.Methods Rat bone marrow-derived imDC were transfected by IKK2dn and loaded by BN antigen,then cultured with Lewis rats T cells in vitro.From these Treg,CD4 + CD25 T cells were screened by magnetic active cell sorting (MACS),then incubated in secondary mixed lymphocyte reaction ( MLR),with Lewis rat T lymphocytes.Results By MACS,the purity of CD4 + CD25 - T cells was more than 95％.The result of secondary MLR displayed the absorbance value was significantly lower in the CD4 + CD25 - T cells group ( 0.106 ± 0.006 ) than that in BN antigen group (0.189 ± 0.007 ),Adv0-CD4 +T cells group (0.419 ± 0.014) and the third donor group (0.200 ± 0.008) (P ＜0.05).Conclusion By MACS sereening,we can obtain high purity of CD4 + CD25-T cells from Treg induced by imDC transfected with IKK2dn and loaded by BN rat antigen and recipient Lewis rats T cells.And these T cells can inhibit T cell proliferation with the donor antigen specificity. Key words: Dendritic cells; IKK2dn; Mixed lymphocyte reaction; Regulatory T cells

Isolation, purification and function of mouse CD4 + CD25+ regulatory T lymphocytes in vitro

Objective To establish a method for isolation and purification of CD4+ CD25+ regulatory T lymphocytes (Treg), and to identify partial functions of these cells. Methods Lymphocytes were isolated from the mouse spleens and then CD4+ CD25+ T cells were sorted by magnetic bead cell sorting (MACS) system. The purity of CD4+ CD25+ T cells were analyzed by flow cytometry (FCM). The activity of them was detected by trypan blue staining. Interleukin (IL)-2 and IL-10 levels in culture supernatant were determined by enzyme linked immunosorbent assay (ELISA). Results The purity of CD4+ CD25+ T cells sorted by MACS was 83%-96%. There was significant difference in the levels of IL-2 and IL-10 secreted by lymphocytes among CD4+ CD25+ Treg group [ (10.25±3.31), (40.32±8.05) ng/L], CD4+ CD25- T group [(58.21±13.05),(11.52±3.01)ng/L] and mixed culture group [ (39.54±13.82), (31.25±4.36)ng/L (P<0.05,P<0.01).Conclusion High purity of CD4+ CD25+ Treg with immune regulatory function can be isolated by MACS. The immunosuppression of CD4+ CD25+ Treg to CD4+ CD25- T was related to the regulation of IL-10 through IL-2. Key words: T cells; CD4; CD25; Immunosuppression

Global DNA Methylation Status of CD4+ T-Cell Population in Mielodysplastic Syndromes.

Abstract 4424 IntroductionRecent studies have demonstrated that sequential administration of demethylating or immunomudulator agents have clinical efficacy in patients with myelodysplastic syndromes (MDS). Demethylating agents induce an optimal re-expression of epigenetically silenced tumor suppressor genes. However, the global DNA demethylation observed in malignant cells during treatment doesn't guarantee a better prognosis, suggesting the presence of others important unknown factors. On the other hand, global DNA hypomethylation of CD4+ T-cells have been related with autoimmune pathology diseases like systemic lupus erythematosus (SLE). The aim of the present study is to establish the degree of the global DNA methylation in CD4+ T-cells in MDS patients and their potential dysfunction. Patients and methodsEight MDS patients with low-risk, according to IPSS (between 0 and 1), diagnosed by cytology, cytogenetics and immunophenotype, and 4 healthy donors have been studied. Peripheral blood mononuclear cells were obtained by ficoll density gradient. Negative CD4+ selection followed by a positive selection were performed using the MACS system (Miltenyi). In patients with CD34+ cells expressing CD4+ antigen, lymphocyte isolation was done using a FACSAria sorter (BD Biosciences). Purity of CD4+ T-cells was higher than 90% in all cases. Cell DNA extraction was carried out with the QIAamp DNA blood mini kit (Qiagen). DNA concentration and 260:280 absorbance ratios were calculated with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). Global DNA methylation content was measured by means of an ELISA using an anti-5-mC mAb (Calbiochem) 1/400 diluted. Before proceeding with patient samples, an optimal DNA concentration to evaluate global methylation levels was established. With this purpose, a reference DNA sample of purified CD4+ T cells (374 ng/ml) was twofold serially diluted in TE (starting at 1:1000 dilution) to observe the kinetics of our ELISA. The best DNA concentration range was 0.025–0.05 ng/ml (Figure 1). Methylation indices (MI) were calculated by getting the ratio between optical density (OD) and DNA concentration for each sample. To minimize experimental variability between plates a reference sample was included in each run. Control and patient MI were corrected by establishing the ratio with the reference MI. ResultsThe global DNA methylation indices obtained by ELISA (Figure 2) displayed that most of MDS patients studied presented a global DNA hypomethylation in CD4+ T-cells, and 3 with IPSS 0 showed and important decrease. ConclusionThese preliminary results showed that there are low-risk MDS patients with hypomethylation in CD4+ T-cells. This observation may suggest an autoimmune component in these malignancies as the one described in SLE. To address this hypothesis we are increasing the number of patients studied to all IPSS categories, to test if this phenomenon is highly represented in MDS. In the other hand, we are studying the presence of autoimmune-related factors, like the expression of integrins adhesive receptors such as LFA-1, in patients showed CD4 T-cells hypomethylation. [Display omitted] [Display omitted] Disclosures:Guerra-Moreno:Celgene: Research Funding. Vallespi:Celgene: Research Funding.

Mast Cells Regulate the Magnitude and the Cytokine Microenvironment of the Contact Hypersensitivity Response

The role that mast cells play during contact hypersensitivity (CS) response is unclear because some studies have shown that mast cell-deficient mice have relatively intact CS responses whereas others have shown opposing results. Mast cells secrete a wide range of immunomodulatory mediators and can potentially influence the type of immune response generated in the skin during CS. Therefore, we examined the type of microenvironment generated during CS in both W/Wv mast cell-deficient and wild-type mice in response to different immunizing doses of hapten (oxazolone). The CS response elicited after low-dose oxazolone was significantly diminished in W/Wv mice compared with wild-type mice. Unexpectedly, the CS response elicited in W/Wv mice immunized with high-dose oxazolone was more severe compared with wild-type mice. In addition, after immunization with high-dose oxazolone, the granulocyte infiltrate in W/Wv mice was increased by twofold compared with wild-type mice. A shift in the cytokine milieu toward the expression of type-1 cytokines as well as a significant increase in the local adhesion of neutrophils and CD4 T cells in the microvasculature of the skin was observed after hapten challenge in W/Wv mice immunized with high-dose oxazolone compared with wild-type mice. These results suggest that mast cells can act as regulators and inducers of the inflammatory response depending on immunizing stimulus strength.

Biological features of intrahepatic CD4+CD25+ T cells in the naturally tolerance of rat liver transplantation

The biological features of intrahepatic CD4(+) CD25(+) T regulatory cells in the naturally tolerance of rat liver transplantation were explored. Orthotopic liver transplantation was performed in two allogeneic rat strain combinations, one with fatal immunosuppression despite a complete major histocompatibility complex mismatch. The subjects were divided into three groups according to different donors and recipients [Tolerance group: LEW-to-DA; Rejection group: DA-to-LEW; Syngegnic group (control group): DA-to-DA]. The proportion of intrahepatic CD4(+)CD25(+) T cells from three groups was determined by flow cytometry (FCM) in different time. The intrahepatic CD4(+)CD25(+) T cells were isolated by magnetic activated cell sorting (MACS) method and identified by FCM. The Foxp3 mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR). And their suppression on the proliferation of CD4(+)CD25(-) T effector cells was analyzed by cell proliferation assay in vitro. Beginning immediately after transplantation, the proportion of Treg cells increased over time in both allogeneic groups but was significantly greater in the Rejection group. The proportion of Treg cells declined after day 5, and such reduction was more dramatic in the Rejection group than in the Tolerance group. Animals in the Tolerance group showed a second increase in the proportion after day 14. Intrahepatic CD4(+) CD25(+) T cells isolated from spontaneous tolerance models inhibited the proliferation of mixed lymphocyte reaction. The purity of CD4(+)CD25(+) T cells sorted by MACS was 86%-93%. The CD4(+)CD25(+) T cells could specifically express the Foxp3 gene compared with CD4(+)CD25(-) T cells. In vitro, the spleen cells from LEW rats can irritate the proliferation of CD4(+)CD25(+) T cells more obviously than the syngegnic spleen cells. CD4(+)CD25(+) Tr cells could suppress the proliferation of CD4(+)CD25(-) T cells, but the inhibition was reversed by exogenous IL-2 (200 U/mL). The CD4(+)CD25(+) T regulatory cells specifically express the Foxp3 gene, which may play an important role in the induction of liver transplantation tolerance by suppressing the reaction of effective T cells.

Isolation and characterization of human CD4+CD25+ T regulatory cells for early phase clinical trials in hematopoietic cell transplantation

Naturally occurring CD4+CD25+ T regulatory cells (Tregs) are a subpopulation of CD4+ T cells vital to homeostasis and maintenance of tolerance. Tregs develop in the thymus and are characterized by expression of the FoxP3 transcription factor. Tregs suppress the proliferation and expansion of conventional CD25-CD4+ and CD8+ T cells through direct cell-cell contact. Defect in Tregs results in multi-organ autoimmune disorders. Adoptive transfer of Tregs cured diabetes in NOD mice and inflammatory bowel disease in murine models. Co-transplantation of Tregs with conventional CD4+ and CD8+ T cells has been shown to control acute graft versus host diseases (GVHD) without abrogating graft versus tumor (GVT) effects in murine bone marrow transplant models.Translating this novel strategy to suppress acute GVHD without loss of GVT holds great promise not only in clinical hematopoietic cell transplantation (HCT), but also in adoptive cellular therapy beyond HCT. Large scale isolation of sufficient human Tregs for therapeutic application remains challenging. Tregs comprise 2–10% of CD4+ T cells. In contrast to rodent Tregs, human CD4+CD25+ cells are more heterogeneous because CD25 is also expressed at lower level by activated non-suppressive conventional T cells and B cells. In addition, immunophenotype of human Tregs is less well studied. Here, we explore the isolation and characterization of human CD4+CD25+ Tregs from peripheral blood mononuclear cells of healthy blood donors and from CD34+ cell depleted fraction of allogenic donors. CD25+ cells were isolated by three different approaches: 1) CD4+ enrichment followed by CD25+ selection, 2) CD19 depletion followed by CD25+ selection, 3) CD25+ selection without prior enrichment or depletion, using magnetic beads. The purities of isolated CD4+CD25+ cells were 95%, 93%, and 93% respectively by FACS. The percentage of CD25 high expressing cells was also similar among the three approaches. More than 90% purity of CD4+CD25+ cells was consistently obtained by CD25+ selection alone using CliniMACS® CD25 microbeads via double column separation utilizing the AutoMACS®. The yield of CD25+ cells varied based upon the ratio of cells/buffer/microbeads. The isolated CD25+ cells were then sorted into CD4+CD25bright (top 25–30%) and CD4+CD25dim (bottom 25–30%) by FACS, fixed and stained for intracellular expression of FoxP3. More than 95% of CD4+CD25bright cells expressed FoxP3. In contrast, only 40% of CD4+CD25dim cells expressed FoxP3. No FoxP3 expression was detected in CD4+CD25− cells. Murine Tregs express a variety of co-stimulatory molecules, however, expression of these molecules is less well characterized on human CD4+CD25+ cells. Using multi-color FACS, we observed that human FoxP3+CD4+CD25bright cells express high levels of CD27 (98%), CD28 (100%), and CD30 (50%) and low levels (<10%) of CTLA-4, GITR, 4-1BB, PD-1 and ICOS on the cell surface. However, CTLA-4, GITR, PD-1 and ICOS were expressed intracellularly in more than 50% of FoxP3+CD4+CD25bright cells. OX40, TRAIL, ICOS ligand and FasL were not detected on the cell surface or in the cytoplasm of FoxP3+CD4+CD25bright cells. FoxP3+CD4+CD25bright cells also express CD62L and CCR5. In summary, highly purified CD4+CD25+ Treg cells can be isolated by magnetic beads and cell sorting. Importantly, the majority of the cells express FoxP3. Utilizing this technique, the clinical application of Tregs appears feasible.

Isolation and Characterization of Human CD4+CD25+ T Regulatory Cells for Early Phase Clinical Trials in Hematopoietic Cell Transplantation.

Naturally occurring CD4+CD25+ T regulatory cells (Tregs) are a subpopulation of CD4+ T cells vital to homeostasis and maintenance of tolerance. Tregs develop in the thymus and are characterized by expression of the FoxP3 transcription factor. Tregs suppress the proliferation and expansion of conventional CD25-CD4+ and CD8+ T cells through direct cell-cell contact. Defect in Tregs results in multi-organ autoimmune disorders. Adoptive transfer of Tregs cured diabetes in NOD mice and inflammatory bowel disease in murine models. Co-transplantation of Tregs with conventional CD4+ and CD8+ T cells has been shown to control acute graft versus host diseases (GVHD) without abrogating graft versus tumor (GVT) effects in murine bone marrow transplant models.Translating this novel strategy to suppress acute GVHD without loss of GVT holds great promise not only in clinical hematopoietic cell transplantation (HCT), but also in adoptive cellular therapy beyond HCT. Large scale isolation of sufficient human Tregs for therapeutic application remains challenging. Tregs comprise 2–10% of CD4+ T cells. In contrast to rodent Tregs, human CD4+CD25+ cells are more heterogeneous because CD25 is also expressed at lower level by activated non-suppressive conventional T cells and B cells. In addition, immunophenotype of human Tregs is less well studied. Here, we explore the isolation and characterization of human CD4+CD25+ Tregs from peripheral blood mononuclear cells of healthy blood donors and from CD34+ cell depleted fraction of allogenic donors. CD25+ cells were isolated by three different approaches: 1) CD4+ enrichment followed by CD25+ selection, 2) CD19 depletion followed by CD25+ selection, 3) CD25+ selection without prior enrichment or depletion, using magnetic beads. The purities of isolated CD4+CD25+ cells were 95%, 93%, and 93% respectively by FACS. The percentage of CD25 high expressing cells was also similar among the three approaches. More than 90% purity of CD4+CD25+ cells was consistently obtained by CD25+ selection alone using CliniMACS® CD25 microbeads via double column separation utilizing the AutoMACS®. The yield of CD25+ cells varied based upon the ratio of cells/buffer/microbeads. The isolated CD25+ cells were then sorted into CD4+CD25bright (top 25–30%) and CD4+CD25dim (bottom 25–30%) by FACS, fixed and stained for intracellular expression of FoxP3. More than 95% of CD4+CD25bright cells expressed FoxP3. In contrast, only 40% of CD4+CD25dim cells expressed FoxP3. No FoxP3 expression was detected in CD4+CD25− cells. Murine Tregs express a variety of co-stimulatory molecules, however, expression of these molecules is less well characterized on human CD4+CD25+ cells. Using multi-color FACS, we observed that human FoxP3+CD4+CD25bright cells express high levels of CD27 (98%), CD28 (100%), and CD30 (50%) and low levels (<10%) of CTLA-4, GITR, 4-1BB, PD-1 and ICOS on the cell surface. However, CTLA-4, GITR, PD-1 and ICOS were expressed intracellularly in more than 50% of FoxP3+CD4+CD25bright cells. OX40, TRAIL, ICOS ligand and FasL were not detected on the cell surface or in the cytoplasm of FoxP3+CD4+CD25bright cells. FoxP3+CD4+CD25bright cells also express CD62L and CCR5. In summary, highly purified CD4+CD25+ Treg cells can be isolated by magnetic beads and cell sorting. Importantly, the majority of the cells express FoxP3. Utilizing this technique, the clinical application of Tregs appears feasible.

Isolation of Human CD4+CD25+ T Cells with Regulatory Capacity for Clinical Trials.

Donor derived CD4+CD25+ regulatory T (Treg) cells do not induce graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT), but actively suppress GVHD induced by CD25− conventional T (Tconv) cells when co-transplanted at a 1:1 ratio in murine model systems. Purified human CD4+CD25+ Treg cells display similar phenotypic and functional characteristics as their murine counterparts, i.e. they constitutively express high levels of intracellular CTLA-4, preferentially express the forkhead/winged helix transcription factor Foxp3, they are anergic to T cell receptor-mediated stimulation, and suppress the proliferation of Tconv cells after polyclonal or allogeneic activation in vitro. Thus, their adoptive transfer is a promising strategy for the prevention of GVHD in patients after allogeneic BMT. For potential clinical applications, efficient protocols for the isolation of CD4+CD25+ Treg cells from peripheral blood are required. In laboratory-scale experiments we determined that depletion of B cells (that partially express CD25), followed by repetitive positive selection of the remaining CD25+ cells with paramagnetic beads, yields a more than 90% pure population of CD4+CD25+ T cells. We now applied this protocol for the large-scale isolation of human CD4+CD25+ T cells from leukapheresis products of healthy volunteers under GMP conditions using the CliniMACS® device. B cell depletion with CD19-beads, followed by three cycles of CD25-enrichment with CD25-coupled microbeads resulted in an average 90% purity of CD4+CD25+ T cells (range 86% to 94%). Positively selected cells constituted on average 1.56% of MNC (range 0.84% to 2.0%), resulting in absolute numbers of 63 to 261 x 106 cells, depending on the initial size of the leukapheresis product. Since T cells with regulatory capacity predominantly reside within the CD4+CD25high subpopulation, we further determined the relative proportion of CD25high to CD25intermediate CD4+ T cells within the cell product. CD4+CD25high T cells represented on average 42% of the purified CD4+CD25+ T cell population (range 26% to 60%) as compared to 10.6% (range 8.5% to 13%) of the CD4+CD25+ T cells within the starting population. CD4+CD25+ T cells isolated according to this GMP protocol expressed higher levels of Foxp3 mRNA and Foxp3 protein as well as higher levels of intracellular CTLA-4 as compared to CD4+CD25− T cells. In addition, they showed functional characteristics of Treg cells as they were hypoproliferative to polyclonal stimulation in vitro and inhibited the proliferation of co-cultured CD4+CD25− T cells in a standard suppression assay. The ability to isolate highly enriched Treg cell populations in sufficient numbers under GMP conditions will accelerate their clinical application in allogeneic BMT.