Carboxyfluorescein Diacetate Succinimidyl Ester Dilution Research Articles

Evaluation of T Cell Proliferation Using CFSE Dilution Assay: A Comparison between Stimulation with PHA and Anti-CD3/Anti-CD28 Coated Beads.

A decrease in T cell count or reduced T cell function can be indicative of T cell immunodeficiency. In the present study, T-cell function was assessed using Carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution test after stimulation with commonly used Phytohaemagglutinin (PHA) or anti-CD3/anti-CD28 coated beads in pediatric patients with recurrent infections. Seven infants with recurrent infections and seven sex/age-matched healthy infants were included in this study. A blood cell count, immunophenotyping, and serum immunoglobulin level were performed. The proliferation of T cells was also assessed with CFSE dilution after stimulation with PHA or anti-CD3/anti-CD28 coated beads. This study showed increased IgA, IgG, and IgM levels in patients compared to the controls. In contrast to the controls, the immunophenotyping results showed a significant decline in the number of CD4+ T cells in patients. Although there was no difference in CD3+ T cell proliferation between patients and controls, the CD4+ and CD8+ T cell proliferation rates were significantly decreased in patients when stimulated with PHA. As a mitogen with the potential for maximum proliferation of T cells, PHA is better able to distinguish between patients with recurrent infections and controls than anti-CD3/anti-CD28, which mimics only the TCR pathway for stimulation of T cells.

IgE recognition of the house dust mite allergen Der p 37 is associated with asthma

House dust mite (HDM) allergens are major elicitors of allergic reactions worldwide. Identification, characterization, and evaluation of diagnostic utility of a new important HDM allergen was performed. A cDNA coding for a new Dermatophagoides pteronyssinus (Dp) allergen, Der p 37, was isolated from a Dp expression library with allergic patients' IgE antibodies. Recombinant Der p 37 (rDer p 37) expressed in Escherichia coli was purified, then characterized by mass spectrometry, circular dichroism, and IgE reactivity by ImmunoCAP ISAC technology with sera from 111 clinically defined HDM-allergic patients. The allergenic activity of rDer p 37 was studied by basophil activation and CD4+ T-cell responses by carboxyfluorescein diacetate succinimidyl ester dilution assays. Specific antibodies raised against rDer p 37 were used for the ultrastructural localization of Der p 37 in mites by immunogold transmission electron microscopy. Der p 37, a 26 kDa allergen with homology to chitin-binding proteins, is immunologically distinct from Der p 15, 18, and 23. It is located in the peritrophic membrane of fecal pellets. Der p 37 reacted with IgE antibodies from a third of HDM-allergic patients and induced specific basophil- and CD4+ T-cell activation. Der p 37 IgE-positive patients had significantly higher IgE levels to major HDM allergens, reacted with more HDM allergens, and had a higher risk (odds ratio=3.1) of asthma compared to Der p 37-negative patients. Der p 37, a new Dp allergen recognized by a third of HDM-allergic patients, may serve as a surrogate marker for severe HDM sensitization and asthma.

Antigen-specific CD4+ and CD8+ T-cell responses in PBMC from pony mares immunized with either native or recombinant zona pellucida vaccines

Few studies have investigated the cell mediated immune response during zona pellucida-based immunocontraception, despite hypothesized cytotoxic T-cell involvement in ovarian dysfunction associated with these vaccines. This study aimed to investigate antigen-specific anamnestic responses of helper (CD4+) and cytotoxic (CD8+) T-lymphocytes in peripheral blood mononuclear cells (PBMC) isolated from pony mares before and after their treatment with native porcine zona pellucida (pZP), recombinant pZP3 and pZP4 antigens (reZP) or adjuvanted saline. Mares were randomly assigned to pZP, reZP and control groups (n = 7 per group). Treatments consisted of a primary vaccination or saline (V1; Day 0) incorporating Freund's modified complete adjuvant, followed by a single booster (V2; Day 35) incorporating Freund's incomplete adjuvant. Peripheral blood mononuclear cells were isolated and cryopreserved immediately prior to V1 (Day 0) and five weeks post V2 (Day 70). Relative proliferation of T-lymphocytes in response to pZP antigen was assessed using carboxyfluorescein diacetate succinimidyl ester dilution with immunophenotyping, analysed via flow cytometry. Significant pZP-specific CD4+ and CD8+ T-lymphocyte responses were detected in PBMC isolated from mares treated with either pZP or reZP, in comparison to pre-treatment samples. In the pZP group, but not the reZP group, CD8+ T-cell proliferation showed significant negative correlations to circulating progesterone, oestradiol and anti-Müllerian hormone levels. Results suggest that antigen-specific CD8+ T-cells may play a role in ovarian suppression observed during pZP immunocontraception in this species.

A Low Effective Dose of Interleukin-7 Is Sufficient to Maintain Cord Blood T Cells Alive without Potentiating Allo-Immune Responses

Slow reconstitution of T cell immunity remains a critical issue after umbilical cord blood (CB) transplantation. Although this may be a consequence of the low cell dose, it may also reflect the propensity of naïve T cells, which predominate in CB, to undergo apoptotic cell death. Exogenous interleukin 7 (IL-7) can prevent apoptosis of naïve T cells, but at high concentrations, IL-7 may also expand alloreactive T cells, thereby aggravating the risk of graft-versus-host disease. We evaluated the survival of CB T cells from 34 healthy full-term pregnancies, and we found wide interdonor variation, from 17.4% to 79.7%, of CB T cells that were still alive after being rested for 4 days in culture medium without cytokine supplementation. The viability of CB T cells was negatively correlated to infant birth weight (Spearman's ρ = .376; P = .031) and positively correlated to venous CB pH (ρ = .397; P = .027); both associations were confirmed by multivariate analysis (P = .023 and P = .005, respectively). A low supplemental concentration (100 pg/mL) of recombinant human IL-7 was sufficient to maintain the viability of cryopreserved/thawed CB T cells, with most (>80%) cells remaining in a quiescent state and without significant changes in their CD4/CD8 ratio and the proportion of CD4+ CD31+ PTK7+ recent thymic emigrants. IL-7 at 100 pg/mL did not lead to any significant enhancement of the alloreactive response of CB T cells, as evaluated by proliferation rates (thymidine incorporation and carboxyfluorescein diacetate succinimidyl ester dilution) and interferon-gamma production (ELISPOT). This effective concentration of IL-7 is far lower than that obtained in vivo after pharmacological administration of the cytokine. This study suggests that administration of lower doses of recombinant human IL-7 than used in previous clinical trials may be sufficient to sustain the viability of infused CB T cells and, thus, help to accelerate naïve T cell reconstitution without potentiating their alloreactivity.

Casitas B Lineage Lymphoma b Is a Key Regulator of Peripheral Tolerance in Systemic Lupus Erythematosus

To analyze whether the expression and modulation of T cell receptor (TCR) signaling is dependent on Casitas B lineage lymphoma b (Cbl-b) in T cells from patients with systemic lupus erythematosus (SLE) upon stimulation with a tolerogenic substance. Peripheral blood mononuclear cells were obtained from 20 patients with SLE (active disease or in remission) and 20 healthy controls. Levels of Cbl-b expression were measured using reverse transcription-polymerase chain reaction and Western blotting in peripheral CD4+ T cells from SLE patients and healthy controls upon anergy induction. Cell proliferation was measured using the carboxyfluorescein diacetate succinimidyl ester dilution method. Cytokine production was analyzed by luminometry, and surface expression of activation markers was assessed by flow cytometry. Transfection assays were performed to induce overexpression of Cbl-b, and phosphorylation of TCR-associated kinases was evaluated. CD4+ T cells from SLE patients displayed resistance to anergy (as evidenced by increased cell proliferation, interleukin-2 production, and expression of activation and costimulatory markers), and this was associated with altered Cbl-b expression. Upon ionomycin treatment, primary T cells showed enhanced MAPK activity and decreased Akt phosphorylation, which was representative of the anergic state. In T cells from lupus patients, Cbl-b overexpression led to increased expression of phosphorylated MAPK, thus indicating the reversibility of anergy resistance. These findings suggest that abnormal peripheral tolerance in SLE is caused by a deficiency in Cbl-b, and that this ubiquitin ligase plays a key role in regulating TCR signaling during the induction of peripheral tolerance.

CD4+CD25+ regulatory T cells from MRL/lpr mice were functionally more active in vitro but did not prevent spontaneous as well as adriamycin-induced nephropathy in vivo

The frequency and function of Tregs are important in the pathogenesis of SLE. Nonetheless, the function of Tregs is still controversial in SLE patients and lupus mouse models. In the present study, we investigated the suppressive function of Tregs from MRL/lpr mice in vitro and in vivo by using an alternative quantitative assay. We assessed the suppressive function of CD4(+)CD25(+) Tregs, the proliferative activity of CD4(+)CD25(-) effector T cells (Teffs) and the feeder activity of CD11c(+) dendritic cells (DCs), isolated from the spleens of MRL/lpr mice and wild-type (WT) MRL/+ mice, by carboxyfluorescein diacetate succinimidyl ester dilution assay stimulated with two distinct types of signals, weak and strong. In order to assess the protective function of Tregs from an immune-mediated disease in vivo, we induced renal damage by injecting adriamycin (ADN) into the mice. The in vitro assay showed enhanced suppressive activity of Tregs and feeder activity of DCs, but far less proliferative activity of Teffs from MRL/lpr mice, compared with those from the WT mice. The in vivo study showed more severe ADN-induced nephropathy in MRL/lpr mice than in the WT mice, while mild interstitial nephritis had already begun spontaneously by 16 weeks in MRL/lpr mice. It was suggested that Tregs from MRL/lpr mice were functionally competent and intrinsically more active in vitro, but they were not capable of preventing the ADN-induced as well as the spontaneously developing nephropathy in vivo.

Reversal of nonstructural protein 3‐specific CD4+ T cell dysfunction in patients with persistent hepatitis C virus infection

Hepatitis C virus (HCV)-specific T cell responses are essential for HCV control, and chronic infection is characterized by functionally altered antigen-specific T cells. It has been proposed that the early inactivation of specific CD4(+) T cell responses may be involved in establishment of HCV persistence. We have investigated whether HCV-specific CD4(+) T cells dysfunction can be reversed in vitro. Nonstructural protein 3 (NS3) and core-specific CD4(+) T cells from eight chronically infected and eight spontaneously resolved HCV individuals were selected through transient CD154 (CD40 ligand) expression, and their functional profile (IFN-γ, IL-2, TNF-α, IL-10 and IL-4 production by enzyme-linked immunospot assay, cytometric bead array and intracellular cytokine staining, and proliferation by carboxy-fluorescein diacetate succinimidyl ester dilution assay) was determined both ex vivo and after in vitro expansion of sorted CD154-expressing cells in the absence of specific antigen in IL-7/IL-15-supplemented medium. Ex vivo bulk CD4(+) T cells from chronic patients expressed CD154 in most cases, albeit at lower frequencies than those of resolved patients (0.11%vs 0.41%; P = 0.01), when stimulated with NS3, but not core, although they had a markedly impaired capacity to produce IL-2 and IFN-γ. Antigen-free in vitro expansion of NS3-specific CD154(+) cells from chronic patients restored IFN-γ and IL-2 production and proliferation to levels similar to those of patients with spontaneously resolved infection. Hence, NS3-specific CD4(+) T cell response can be rescued in most chronic HCV patients by in vitro expansion in the absence of HCV-specific antigen. These results might provide a rationale for adoptive immunotherapy.

Detrimental Effect of the Proteasome Inhibitor, Bortezomib in Bacterial Superantigen- and Lipopolysaccharide-induced Systemic Inflammation

Bacterial superantigen (BSAg)-induced toxic shock syndrome (TSS) and bacterial lipopolysaccharide (LPS)-induced shock are characterized by severe systemic inflammation. As nuclear factor kappaB (NF kappaB) plays an important role in inflammation and bortezomib, a proteasome inhibitor widely used in cancer chemotherapy, is a potent inhibitor of NF kappaB activation, we evaluated the therapeutic and prophylactic use of bortezomib in these conditions using murine models. Bortezomib prophylaxis significantly reduced serum levels of many cytokines and chemokines induced by BSAg. However, at 3 hours, serum level of TNF-a, an important cytokine implicated in TSS, was significantly reduced but not abolished. At 6 hours, there was no difference in the serum TNF-a levels between bortezomib treated and untreated mice challenged with staphylococcal enterotoxin B (SEB). Paradoxically, all mice treated with bortezomib either before or after BSAg challenge succumbed to TSS. Neither bortezomib nor BSAg was lethal if given alone. Serum biochemical parameters and histopathological findings suggested acute liver failure as the possible cause of mortality. Liver tissue from SEB-challenged mice treated with bortezomib showed a significant reduction in NF kappaB activation. Because NF kappaB-dependent antiapoptotic pathways protect hepatocytes from TNF-alpha-induced cell death, inhibition of NF kappaB brought forth by bortezomib in the face of elevated TNF-alpha levels caused by BSAg or LPS is detrimental.

High frequencies of less differentiated and more proliferative WT1‐specific CD8+ T cells in bone marrow in tumor‐bearing patients: An important role of bone marrow as a secondary lymphoid organ

In tumor-bearing patients, tumor-associated antigen (TAA)-specific CTLs are spontaneously induced as a result of immune response to TAAs and play an important role in anti-tumor immunity. Wilms' tumor gene 1 (WT1) is overexpressed in various types of tumor and WT1 protein is a promising pan-TAA because of its high immunogenicity. In this study, to clarify the immune response to the WT1 antigen, WT1-specific CD8(+) T cells that were spontaneously induced in patients with solid tumor were comparatively analyzed in both bone marrow (BM) and peripheral blood (PB). WT1-specific CD8(+) T cells more frequently existed in BM than in PB, whereas frequencies of naïve (CCR7(+) CD45RA(+)), central memory (CCR7(+) CD45RA-), effector-memory (CCR7- CD45RA(-)), and effector (CCR7- CD45RA(+)) subsets were not significantly different between BM and PB. However, analysis of these subsets for the expression of CD57 and CD28, which were associated with differentiation, revealed that effector-memory and effector subsets of the WT1-specific CD8(+) T cells in BM had less differentiated phenotypes and more proliferative potential than those in PB. Furthermore, CD107a/b functional assay for WT1 peptide-specific cytotoxic potential and carboxyfluorescein diacetate succinimidyl ester dilution assay for WT1 peptide-specific proliferation also showed that WT1-specific CD8(+) T cells in BM were less cytotoxic and more proliferative in response to WT1 peptide than those in PB. These results implied that BM played an important role as a secondary lymphoid organ in tumor-bearing patients. Preferential residence of WT1-specific CD8(+) T cells in BM could be, at least in part, explained by higher expression of chemokine receptor CCR5, whose ligand was expressed on BM fibroblasts on the WT1-specific CD8(+) T cells in BM, compared to those in PB. These results should provide us with an insight into WT1-specific immune response in tumor-bearing patients and give us an idea of enhancement of clinical response in WT1 protein-targeted immunotherapy.

Cutting Edge: Immunosuppressant as Adjuvant for Tolerogenic Immunization

Kang Y, Xu L, Wang B, Chen A, Zheng G. J Immunol. 2008;180(8):5172–5176 PURPOSE OF THE STUDY. Immunosuppressive agents are used frequently for the treatment of allergy, autoimmune disease, and transplant rejection but are thought to provide only temporary benefit. The authors of this study sought to determine if dexamethasone, a glucocorticoid with potent immunosuppressive properties, could promote long-term antigen-specific tolerance when administered with a peptide immunogen. METHODS. The authors used a model of delayed-type hypersensitivity (DTH) to hen ovalbumin by subcutaneously injecting ovalbumin twice during a 2-week interval into BALB/c mice. Mice with established DTH to ovalbumin were then immunized with an ovalbumin-derived MHCII-restricted peptide in the presence or absence of dexamethasone. All mice were retested for DTH at 2-week and 4- to 5-month time points after completion of immunization by injecting ovalbumin into the footpad and measuring the increase in footpad thickness. Blood-derived CD4+CD25+Foxp3+ regulatory T cells (Tregs) were quantitated by labeling peripheral white blood cells with carboxyfluorescein diacetate succinimidyl ester (CFSE), stimulating the cultures with ovalbumin peptide, and analyzing for CFSE dilution (indicating cell division). Dendritic cells (DCs) in draining lymph nodes (LNs) were assessed for maturity by staining for CD11c, CD83, and CD86, as well as interleukin 10 (IL-10) in some experiments. Transgenic mice containing large numbers of ovalbumin-specific CD4+ T cells were studied also. A similar set of experiments was performed with nonobese-diabetic mice, a mouse model of autoimmune diabetes, by using dexamethasone and an insulin-derived MHCII-restricted peptide. Primary outcomes measured were median time to development of diabetes (as defined by glycosuria) and measurement of Treg numbers and antigen-specific proliferation. RESULTS. Treatment with dexamethasone induced long-term desensitization (as long as 4–5 months) in mice with preestablished DTH to hen ovalbumin as evidenced by a decrease in foot-pad swelling after ovalbumin challenge. This finding was accompanied by an expansion of CD4+CD25+Foxp3+ Tregs, which largely had specificity for ovalbumin. Dexamethasone, in the presence of ovalbumin, seemed to block DC maturation in draining LNs and facilitated differentiation of IL-10+ DCs. Furthermore, treatment of nonobese-diabetic mice with dexamethasone hindered development of spontaneous diabetes and was associated with the development of long-term antigen-specific persistent Tregs. CONCLUSIONS. Dexamethasone, when applied together with peptide, can promote long-term tolerance. The underlying mechanism may involve dexamethasone's effect on inhibiting DC maturation and promoting development of persistent antigen-specific Tregs. REVIEWER COMMENTS. Development of long-term tolerance, as opposed to transient desensitization, is the end goal of immunotherapy protocols, and this study suggests that glucocorticoids may be effective adjuvants in achieving this goal. The authors of this article speculated that other small-molecule immunosuppressant drugs, including cyclosporine and FK506, may also be effective for this purpose, but there is some evidence that these agents may actually promote development of allergic disease in children after solid organ transplantation.

CD11c + Dendritic Cells Maintain Antigen Processing, Presentation Capabilities, and CD4 + T-Cell Priming Efficacy Under Hypercholesterolemic Conditions Associated With Atherosclerosis

Recent reports suggest dyslipidemia impairs dendritic cell (DC) function and adaptive immunity. This study aimed to characterize the effect of hypercholesterolemia on antigen-presenting cell function of DCs and DC-dependent CD4(+) T-cell responses. DCs incubated in vitro with acetylated low-density lipoprotein cholesterol with or without an acyl-coenzyme A:cholesterol acyl-transferase inhibitor maintained their ability to prime CD4(+) T cells. Analysis of T-cell proliferation and interferon-gamma and tumor necrosis factor-alpha production after ex vivo coculture of naïve CD4(+) T cells with splenic, inguinal, or iliac DCs from low-density lipoprotein receptor-deficient (LDLR(-/-)) or apolipoprotein E-deficient (ApoE(-/-)) mice fed an atherogenic diet highlighted DC efficacy in effector T-cell generation under hypercholesterolemic conditions. Adoptive transfer of carboxyfluorescein diacetate, succinimidyl ester (CFSE)-labeled naïve CD4(+) T cells in LDLR(-/-) recipients and subsequent immunization demonstrated effective priming of naïve T cells in hypercholesterolemic mice. CFSE dilution analyses revealed that hypercholesterolemic DCs were equipotent in naïve CD4(+) T-cell priming efficacy with normocholesterolemic DCs. Quantitative real-time PCR and flow cytometric analyses demonstrated that DC expression of multiple molecules involved in antigen processing, presentation, and T-cell stimulation remained unaltered by dyslipidemia. Finally, endogenous antigen-primed CD4(+) T cells responded equivalently to a secondary ex vivo antigenic challenge, regardless of whether they were primed in vivo under hypercholesterolemic or control conditions, demonstrating that all essential steps in CD4(+) T-cell responses remain intact under atherogenic conditions. This study affirms that the adaptive immune response prevails under the hypercholesterolemic conditions present in atherosclerosis. In particular, DCs remain functional antigen-presenting cells and maintain their ability to prime CD4(+) T cells even when cholesterol-loaded.

Profound inhibition of antigen-specific T-cell effector functions by dasatinib.

The dual BCR-ABL/SRC kinase inhibitor dasatinib entered the clinic for the treatment of chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia. Because SRC kinases are known to play an important role in physiologic T-cell activation, we analyzed the immunobiological effects of dasatinib on T-cell function. The effect of dasatinib on multiple T-cell effector functions was examined at clinically relevant doses (1-100 nmol/L); the promiscuous tyrosine kinase inhibitor staurosporine was used as a comparator. Purified human CD3+ cells and virus-specific CD8+ T cells from healthy blood donors were studied directly ex vivo; antigen-specific effects were confirmed in defined T-cell clones. Functional outcomes included cytokine production (interleukin-2, IFN gamma, and tumor necrosis factor alpha), degranulation (CD107a/b mobilization), activation (CD69 up-regulation), proliferation (carboxyfluorescein diacetate succinimidyl ester dilution), apoptosis/necrosis induction, and signal transduction. Both dasatinib and staurosporine inhibited T-cell activation, proliferation, cytokine production, and degranulation in a dose-dependent manner. Mechanistically, this was mediated by the blockade of early signal transduction events and was not due to loss of T-cell viability. Overall, CD4+ T cells seemed to be more sensitive to these effects than CD8+ T cells, and naïve T cells more sensitive than memory T-cell subsets. The inhibitory effects of dasatinib were so profound that all T-cell effector functions were shut down at therapeutically relevant concentrations. These findings indicate that caution is warranted with use of this drug in the clinical setting and provide a rationale to explore the potential of dasatinib as an immunosuppressant in the fields of transplantation and T-cell-driven autoimmune diseases.

In vivo and in vitro techniques for comparative study of antiviral T-cell responses in the amphibian Xenopus

Activation of lymphocytes in mammals is often quantified by measuring the amount of proliferation during the expansion phase of an immune response. Bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assays are some of the techniques widely used in mammalian studies of pathogen-induced proliferation and provide a convenient way of quantifying the cellular response. We have extended the use of these proliferation assays to the amphibian Xenopus laevis. We have developed this species as a valuable comparative model to study immunity against a well-known amphibian pathogen, Frog Virus 3 (FV3). Fluorescence activated cell sorting was used to assess the level of BrdU incorporation of lymphocytes in vivo and CFSE dilution in an in vitro activation assay. Both techniques have shown that splenic lymphocytes proliferate specifically upon FV3 challenge. This indicates that common methods for detection of proliferation upon immunologic challenge are easily applied to other vertebrate species, as it highlights the evolutionary conservation of the proliferative nature of immune responses throughout vertebrate phyla.

Tracking cell proliferation using the far red fluorescent dye SNARF‐1

The [(3)H]thymidine incorporation assay and staining of living cells with fluorescent dyes like carboxyfluorescein diacetate, succinimidyl ester (CFSE) have evolved as valuable methods for studying T cell responses. To assess proliferation of cells already labeled by FITC, CFSE, GFP, or other "green" molecules or to simultaneously track two otherwise indistinguishable cell populations in mixed cell cultures, it would be desirable to have a dye with distinct fluorescent properties for this application. We analyzed the dilution of the far red fluorescent dye SNARF-1 in proliferating cells by flow cytometric analysis. The results were compared with the CFSE dilution technique as well as the [(3)H]thymidine incorporation assay. Staining of primary human lymphocytes revealed that SNARF-1 labeling was equivalent to CFSE for estimating proportions of proliferating cells in stimulated cell cultures and yielded results comparable to [(3)H]thymidine incorporation. We showed that SNARF-1 offers the possibility to simultaneously analyze the proliferation of phenotypically indistinguishable subsets of hematopoietic cells and can also be used to track uniformly proliferating, non hematopoietic cells like HEK293. In summary, we have demonstrated that labeling of cells with SNARF-1 allows for estimating cell proliferation of cells of hematopoietic and non-hematopoietic origin.