Expansion of different subpopulations of CD26-/low T cells in allergic and non-allergic asthmatics.

CD26 displays variable levels between effector (TH17 ≫ TH1 > TH2 > Treg) and naïve/memory (memory > naïve) CD4+ T lymphocytes. Besides, IL-6/IL-6R is associated with TH17-differentiation and asthma severity. Allergic/atopic asthma (AA) is dominated by TH2 responses, while TH17 immunity might either modulate the TH2-dependent inflammation in AA or be an important mechanism boosting non-allergic asthma (NAA). Therefore, in this work we have compared the expression of CD26 and CD126 (IL-6Rα) in lymphocytes from different groups of donors: allergic (AA) and non-allergic (NAA) asthma, rhinitis, and healthy subjects. For this purpose, flow cytometry, haematological/biochemical, and in vitro proliferation assays were performed. Our results show a strong CD26-CD126 correlation and an over-representation of CD26- subsets with a highly-differentiated effector phenotype in AA (CD4+CD26-/low T cells) and NAA (CD4-CD26- γδ-T cells). In addition, we found that circulating levels of CD26 (sCD26) were reduced in both AA and NAA, while loss of CD126 expression on different leukocytes correlated with higher disease severity. Finally, selective inhibition of CD26-mRNA translation led to enhanced T cell proliferation in vitro. These findings support that CD26 down-modulation could play a role in facilitating the expansion of highly-differentiated effector T cell subsets in asthma.

CD26 and Asthma: a Comprehensive Review.

Asthma is a heterogeneous and chronic inflammatory family of disorders of the airways with increasing prevalence that results in recurrent and reversible bronchial obstruction and expiratory airflow limitation. These diseases arise from the interaction between environmental and genetic factors, which collaborate to cause increased susceptibility and severity. Many asthma susceptibility genes are linked to the immune system or encode enzymes like metalloproteases (e.g., ADAM-33) or serine proteases. The S9 family of serine proteases (prolyl oligopeptidases) is capable to process peptide bonds adjacent to proline, a kind of cleavage-resistant peptide bonds present in many growth factors, chemokines or cytokines that are important for asthma. Curiously, two serine proteases within the S9 family encoded by genes located on chromosome 2 appear to have a role in asthma: CD26/dipeptidyl peptidase 4 (DPP4) and DPP10. The aim of this review is to summarize the current knowledge about CD26 and to provide a structured overview of the numerous functions and implications that this versatile enzyme could have in this disease, especially after the detection of some secondary effects (e.g., viral nasopharyngitis) in type II diabetes mellitus patients (a subset with a certain risk of developing obesity-related asthma) upon CD26 inhibitory therapy.

CD26: a negative selection marker for human Treg cells.

A major obstacle hampering the therapeutic application of regulatory T (Treg) cells is the lack of suitable extracellular markers, which complicates their identification/isolation. Treg cells are normally isolated via CD25 (IL-2Rα) targeting, but this protein is also expressed by activated CD4(+) effector T (Teff) lymphocytes. Other extracellular (positive or negative) Treg selection markers (e.g., HLA-DR, CD127) are also nonspecific. CD26 is an extracellular peptidase whose high expression has been traditionally used as an indicator of immune activation and effector functions in T cells. Now, we provide flow cytometry data showing high levels of CD26 within CD4(+)CD25(-) or CD4(+)FoxP3(-/low) effector T (Teff) lymphocytes, but negative or low levels (CD26(-/low)) in Treg cells selected according to the CD4(+)CD25(high) or the CD4(+)FoxP3(high) phenotype. Unlike the negative marker CD127 (IL-7Rα), which is down modulated in CD4(+) Teff lymphocytes after TCR triggering, most of these cells upregulate CD26 and take a CD4(+)CD25(+/high) CD26(+) phenotype upon activation. In contrast, there is only a slight upregulation within Treg cells (CD4(+)CD25(high) CD26(-/low)). Thus, differences in CD26 levels between Treg and Teff subsets are stable, and assessment of this marker, in combination with others like CD25, FoxP3, or CD127, may be useful during the quantitative evaluation or the isolation of Treg cells in samples containing activated Teff lymphocytes (e.g., from patients with autoimmune/inflammatory diseases).

Application of thiophilic chromatography to deplete serum immunoglobulins in sample preparation for bidimensional electrophoresis.

Serum is a typical sample for non-invasive studies in clinical research. Its proteome characterization is challenging, since requires extensive protein depletion. Methods used nowadays for removal of high-abundance proteins are expensive or show quite often a low loading capacity, which has strong repercussions on the number of samples and replicates per analysis. In order to deplete immunoglobulins (Igs) and albumin (HSA) from 1 mL serum samples, we have developed a protocol based on a combination of thiophilic chromatography, not previously used in clinical proteomics, and a HSA-specific resin. Ig/HSA-depleted samples, immunoglobulinome and albuminone were analyzed by 2-DE. Thiophilic chromatography, coupled with HSA-depletion, allows a good 2-DE resolution as well as the visualization of new spots. Moreover, it yields enough protein to evaluate technical variability and facilitate subsequent protein identification. To validate the protocol, we carried out a preliminary comparative study between triplicate Igs/HSA-depleted serum samples from healthy control individuals and recently diagnosed/untreated rheumatoid arthritis (RA) patients. RA patients showed several acute phase proteins, as well as additional serum proteins, differentially and significantly regulated. Therefore, thiophilic chromatography can be used as an efficient and economical method in 2-DE to deplete immunoglobulins from large human serum samples before a more extensive fractioning.

On the origin of serum CD26 and its altered concentration in cancer patients.

Dipeptidyl peptidase IV (DPP-IV), assigned to the CD26 cluster, is expressed on epithelial cells and lymphocytes and is a multifunctional or pleiotropic protein. Its peptidase activity causes degradation of many biologically active peptides, e.g. some incretins secreted by the enteroendocrine system. DPP-IV has, therefore, become a novel therapeutic target for inhibitors that extend endogenously produced insulin half-life in diabetics, and several reviews have appeared in recent months concerning the clinical significance of CD26/DPP-IV. Biological fluids contain relatively high levels of soluble CD26 (sCD26). The physiological role of sCD26 and its relation, if any, to CD26 functions, remain poorly understood because whether the process for CD26 secretion and/or shedding from cell membranes is regulated or not is not known. Liver epithelium and lymphocytes are often cited as the most likely source of sCD26. It is important to establish which tissue or organ is the protein source as well as the circumstances that can provoke an abnormal presence/absence or altered levels in many diseases including cancer, so that sCD26 can be validated as a clinical marker or a therapeutic target. For example, we have previously reported low levels of sCD26 in the blood of colorectal cancer patients, which indicated the potential usefulness of the protein as a biomarker for this cancer in early diagnosis, monitoring and prognosis. Through this review, we envisage a role for sCD26 and the alteration of normal peptidase capacity (in clipping enteroendocrine or other peptides) in the complex crosstalk between the lymphoid lineage and, at least, some malignant tumours.

IL-12-dependent activation of ERK1/2 in human T lymphoblasts.

According to some authors, membrane compartmentalization is a key regulator of CD45 function. Indeed, it has been described that CD45 repositioning from raft microdomains to phospholipid-rich plasma membrane areas leads to the activation of extracellular signal-regulated kinases (ERKs). We have previously shown that interleukin-12 (IL-12) increases the expression of CD26, promoting the interaction of CD26 with CD45R0 (a CD45 isoform) and removing CD45R0 from lipid rafts. Thus, this IL-12-dependent removal of CD45RO from rafts could, hypothetically, fulfill functions like the activation of the ERK1/2 pathway. IL-12 is an important interleukin for T cells. Upon interaction with its receptor (interleukin-12 receptor; IL-12R), this cytokine triggers a signalling cascade, where the classical Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway and other additional routes participate. Due to the promitogenic effect of IL-12 and the influence of this cytokine on CD45RO compartmentalization, ERK kinases were likely candidates to be downstream of IL-12R. However, several research groups have rejected a role for these kinases. Now, results in this paper show that the IL-12R binding, similar to the stimulation via T cell receptor (TCR), promotes the activation of the Raf/MEK-1/ERK1/2 pathway. In addition, the IL-12R-associated Janus kinase JAK2, but not TYK2, seems upstream of this important pathway for the proliferation of human T cells. However, even though c-Myc is slightly up-regulated by IL-12 and partially mediates the proliferative effect of IL-12, this transcription factor was not found downstream of ERK1/2.

Interactions between DMPC liposomes and the serum blood proteins HSA and IgG.

The interaction between two serum blood proteins, namely human serum albumin (HSA) and human immunoglobulin G (IgG), with 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) liposomes has been studied in detail using dynamic light scattering, flow cytometry, enzyme-linked immunosorbent assay (ELISA), electrophoretic mobility, differential scanning calorimetry (DSC), and surface tension measurements. HSA and IgG interact with liposomes forming molecular aggregates that remain stable at protein concentrations beyond those of total liposome coverage. Both HSA and IgG penetrate into the liposome bilayer. An ELISA assay indicates that the Fc region of IgG is the one that is immersed in the DMPC membrane. The liposome-protein interaction is mainly of electrostatic nature, but an important hydrophobic contribution is also present.

Differential distribution of both IL-12Rbeta chains in the plasma membrane of human T cells.

IL-12 is a cytokine that stimulates the expression of CD26, a T cell- and raft-associated ectopeptidase. IL-12 also enhances the interaction between CD26 and CD45RO, which removes the phosphatase CD45RO from raft microdomains. Since Janus kinases are known CD45 substrates, our hypothesis was that this relocation of CD45RO in nonraft areas of the membrane could be important to switch off the signaling via cytokine receptors, e.g., the IL-12 receptor (IL-12R). Accordingly, both IL-12R and CD45RO should be equally positioned in the cell membrane upon IL-12R ligation. However, there were no data available on the membrane distribution of IL-12R on human T cells. Working with phytohemagglutinin (PHA) lymphoblasts, we tried to fill that gap. The high-affinity IL-12R is made of two chains: IL-12Rbeta1 and IL-12Rbeta2. Using flow cytometry, Western blot and confocal microscopy, we obtained data suggesting that IL-12Rbeta1 is mainly associated to phospholipid-rich membrane areas, a location even enhanced upon IL-12 incubation of PHA blasts. Instead, IL-12Rbeta2 is found more segregated into membrane rafts, which could explain why two IL-12-triggered events, T-cell proliferation and ERK1/2 activation, are both methyl-beta-cyclodextrin-sensitive events. Ligation of IL-12R with IL-12 seems to induce a partial enrichment of IL-12Rbeta2 in phospholipid-rich areas, where according to our data IL-12Rbeta1 is already present. Therefore, although new data will be required, the present results support the initial hypothesis.

The influence of sodium perfluorooctanoate on the conformational transitions of human immunoglobulin.

In the field of bioscience, the study of the interactions between blood proteins and fluorinated materials is very important from both theoretical and applied points of view. Fluorinated materials have potential use in drug delivery, as blood substitutes, and in biotechnology. Using a combination of ultraviolet-visible (UV-vis) and ultraviolet-circular dichroism (UV-CD) spectroscopies and ion-selective electrodes, the complete interaction of sodium perfluorooctanoate (SPFO) and the most important immunoglobulin (on a quantitative basis) in human serum, immunoglobulin G (IgG), has been evaluated. The study has been focused on bulk solution. By the application of an SPFO selective electrode, it was determined that there were true specific unions between surfactant molecules and IgG structure. The experimental data were presented as Koltz and Scatchard plots and analyzed on the basis of an empirical Hill equation. The conformational changes at the bulk solution were well characterized by UV-vis and UV-CD spectroscopies. As a consequence of these changes, the protein structure was affected.

Prothymosin alpha-receptor associates with lipid rafts in PHA-stimulated lymphocytes.

Lipid rafts are specialized plasma membrane microdomains in which glycosphingolipids and cholesterol are major structural components. Their relative insolubility to nonionic detergents is the most widely used method to purify these structures. Several signalling proteins are associated with these microdomains in T lymphocytes, including receptors for growth factors and cytokines. ProTalpha is a highly conserved and widely distributed protein whose physiological functions remain elusive. In previous works we identified, by means of affinity cross-linking, affinity chromatography and fluorescence microscopy, a set of binding proteins for ProTalpha in human lymphoblasts. Now, this work goes deeply in that ProTalpha receptor description revealing, by different experimental approaches, its presence in lipid rafts. Moreover, our results fit a model in which a tyrosine phosphorylation signalling cascade confined to rafts is initiated upon ProTalpha receptor recognition, which represents an important and promising finding in the research for elucidating the molecular mechanisms underlying the immunomodulatory functions of ProTalpha.

A role for interleukin-12 in the regulation of T cell plasma membrane compartmentation.

The immunological synapse initiates the clustering and stabilization of the T cell receptor by the formation of a large lipid microdomain that accumulates (e.g. CD4/CD8) and segregates (e.g. CD45 and LFA-1) some proteins of the T cell plasma membrane. This work shows that a fraction of transmembrane glycoproteins CD26 and CD45 (the R0 isoform in particular) is present in the rafts of fresh and activated human T lymphocytes. CD26 is proposed as the costimulator of TCR-dependent activation, and CD45 is essential to the T cell activation process because it dephosphorylates at least the inhibitory site of Src kinases. These findings support a more complex model of compartmentation, depending on the stage of T cell maturation and post-transcriptional and post-translational regulation. In addition, interleukin 12 (IL-12; inducer of TH1 responses) drives CD26 and CD45R0 to particular microdomains, thereby involving interleukins in the rules governing raft inclusion or exclusion. The physical association of CD26 and CD45R0 has long been reported. The results presented in this work fit a model in which IL-12 up-regulates a certain type of CD26 expression that interacts on the cell surface with CD45R0, near but outside of the raft core. The use of antisense oligonucleotides for the CD26 mRNAs demonstrated that both events (enhanced by IL-12), CD26-CD45R0 association and membrane compartment redistribution, are related. Thus, CD26 could be part of a shuttling mechanism for CD45 that regulates membrane tyrosine-phosphatase activities, e.g. to control IL-12 receptor-dependent signal transduction.

Clinical interest of the combined use of serum CD26 and alpha-L-fucosidase in the early diagnosis of colorectal cancer.

The purpose of this study was to assess if the combination of CD26 and alpha-L-fucosidase has a role in the diagnosis of colorectal cancer, paying particular attention to the stages in which the tumour is not yet disseminated. CD26 concentration and alpha-L-fucosidase activity were determined in sera from 110 colorectal cancer patients and 46 donors. The combination of CD26 and alpha-L-fucosidase showed a specificity of 100% with a sensitivity of 64% in the diagnosis of colorectal cancer. Interestingly, the combination of both markers had a sensitivity of 75% in the stage I at the highest specificity (100%), providing also high sensitivity levels for the other non-disseminated stages (66% for stages II and III). In conclusion, the combined use of CD26 and alpha-L-fucosidase offers high sensitivity with high specificity in the diagnosis of colorectal cancer, especially at the earliest stage (TNM I).

Interleukin-dependent modulation of HLA-DR expression on CD4and CD8 activated T cells.

Summary Interleukins (IL) regulate different T-cell surface Ag known as activation markers that have distinct functional roles. In this paper, while studying the influence of some cytokines(IL-12, IL-2 and IL-4) on the expression of several markers [CD69,CD25, CD26, CD3, human leukocyte antigen (HLA-DR), CD45R0] in in vitro activated human T lymphocytes, we observed two groups of donors responding to phytohaemagglutinin (PHA) activation with high or low HLA-DRAg expression. We also found that CD4 and CD8 populations had different HLA-DR densities under PHA activation (particularly the high HLA-DR-expressing group). Interleukins, in a dose-dependent manner (IL-2 partially),upregulated these HLA-DR levels. In 5 day cultures, IL-12 and IL-2 enhanced the CD8/CD4 ratio of activated T cells,which was responsible, in part, for the IL-dependent HLA-DR upregulation.IL-12 and IL-2 also upregulated the HLA-DR expression at the molecular level on CD8, and IL-12 downregulated it on CD4 cells. It seems that IL-4 upregulated HLA-DR by shortening the mitogen-dependent regulation kinetics. We hypothesize that the different effect of each IL on HLA-DR expression might be related to the regulation of the dose of antigenic peptide presentation and, thus, also influence TH1/TH2 dominance.