Telomere length of in vivo expanded CD4+CD25+ regulatory T-cells is preserved in cancer patients

Purpose: CD4⁺CD25⁺ regulatory T-cells (Treg) are increased in the peripheral blood of cancer patients. It remains unclear whether this is due to redistribution or active proliferation. The latter would require the upregulation of telomerase activity, whose regulation also remains unknown for Treg. Experimental Design: Treg and CD4⁺CD25⁻ T-cells were isolated from peripheral blood of cancer patients (n=23) and healthy age-matched controls (n=17) and analyzed for their content of T-cell receptor excision circles (TREC) and for telomere length using flow-FISH, real-time PCR and Southern blotting. The in vitro regulation of telomerase of Treg was studied using PCR-ELISA in bulk cultures as well as in isolated proliferating and non-proliferating Treg. Results: Treg isolated from peripheral blood of cancer patients exhibit significantly decreased levels of TREC when compared to Treg from healthy controls. Despite their in vivo proliferation, telomere length is not further shortened in Treg from cancer patients. Accordingly, telomerase activity of Treg was readily inducible in vitro. Notably, sorting of in vitro proliferating Treg revealed a significant telomere shortening in Treg with high-proliferative capacity. The latter are characterized by shortened telomeres despite high telomerase activity. Conclusions: Increased frequencies of Treg in peripheral blood of cancer patients are due to active proliferation rather than due to redistribution from other compartments (i.e., secondary lymphoid organs or bone marrow). In vivo expansion does not further shorten telomere length, probably due to induction of telomerase activity. In contrast, under conditions of strong in vitro stimulation telomerase induction seems to be insufficient to avoid progressive telomere shortening.Herbert Tilg and Anna M. Wolf share senior authorship     

Circulating cytotoxic CD8+ CD28- T cells in ankylosing spondylitis

Circulating CD8⁺ CD28^- T cells were found to be expanded more in patients with ankylosing spondylitis than in an age-matched healthy population (41.2 ± 17.7% versus 18.6 ± 7.6%). The level of CD8⁺CD28^- T cells was dependent on the disease status, but was independent of age. Most of the CD8⁺ CD28^- T cells produced perforin after stimulation in vitro, in contrast to their CD8⁺CD28⁺ counterparts. From the clinical perspective, the percentage of the cytotoxic CD8⁺ CD28^- T cells reflected a more severe course of disease, as it correlated with distinct movement restrictions, as well as the metrology score summarizing cervical rotation (in sitting position), chin-to-jugulum distance, thoracic Schober, chest expansion, and fingers-to-floor distance (P = 0.032).     

EGFRvIII-targeted immunotoxin induces antitumor immunity that is inhibited in the absence of CD4+ and CD8+ T cells

Purpose Immunotoxins as anti-cancer therapeutics have several potential advantages over conventional agents including a high specificity, extraordinary potency, and a lack of an identified mechanism for resistance. It has been clearly demonstrated that Pseudomonas-based immunotoxins have a direct cytotoxic effect. However, delayed and often dramatic antitumor responses seen in human studies with targeted toxins led us to hypothesize that immunologic responses may be a secondary mechanism that enhances the therapeutic efficacy of these novel drugs. Experimental design This hypothesis was tested in a murine system using an immunotoxin, MR1-1 [MR1-1(dsFv)-PE38KDEL], that targets a syngeneic murine homologue of the tumor-specific human epidermal growth factor mutation, EGFRvIII, expressed on a murine cell line. Results Intratumoral treatment with MR1-1 eliminated EGFRvIII-expressing tumors (P < 0.0001). The antitumor activity of MR1-1 was dependent on the expression of EGFRvIII on some, but not all tumors cells, and was significantly inhibited in the absence of CD4+ (P = 0.0193) and CD8+ (P = 0.0193) T cells. MR1-1 induced EGFRvIII-specific immunity (P < 0.0005) and produced long lasting immunity against tumors expressing EGFRvIII as well as EGFRvIII-negative tumors. Conclusions These data suggest that immunotoxins may not be strictly dependent on direct cytotoxicity for their efficacy, but may also be potent inducers of antitumor immunity active even against cells that do not express the targeted antigen.     

The CD4+CD28null and the regulatory CD4+CD25High T-cell phenotypes in patients with ulcerative colitis during active and quiescent disease, and following colectomy

The CD4⁺CD25^High T-cell phenotype has an essential immunoregulatory role, while the CD4⁺CD28^null T-cell reflects immune pathology. We investigated the profiles of the CD4⁺CD25^High and the CD4⁺CD28^null T-cell phenotypes in patients with ulcerative colitis (UC) during active and quiescent phases as well as following colectomy. Fifty-nine UC patients, 34 active (UCa) and 25 quiescent (UCq) together with 19 healthy controls (HC) were included. Ten of 34 UCa patients underwent colectomy due to unremitting UC (UCo). Immunohistochemical phenotypic of the peripheral blood lymphocytes bearing CD4, CD25 or CD28 was done for analyzes by a multiparameter fluorescence activated cell sorting technique. The expression of the CD4⁺CD25^High phenotype was higher in UCq (P < 0.01) or UCo (P < 0.01) group vs UCa group. Further, the expression of the CD4⁺CD28^null phenotype in UCa or UCo group was higher than in the HC group (P < 0.05). However, the expression of the CD4⁺CD28^null phenotype up to 12 months after colectomy was not significantly different from the levels in the same patients during acute phase. Our impression is that a high CD4⁺CD25^High T-cell reflects alleviation of inflammation, while the expression of the CD4⁺CD28^null T-cell phenotype is an etiologic feature in UC patients, and is maintained after removing the affected colon.     

Adoptive transfer of CD3+ T cells and CD4+CD44high memory T cells induces autoimmune pancreatitis in MRL/MpJ mice

The immunopathogenesis of autoimmune pancreatitis (AIP) is poorly understood. Here, we have used MRL/MpJ mice, a model of spontaneous AIP, to address the role of cellular autoimmune processes in the initiation and progression of the disease. Therefore, different T cell subpopulations were adoptively transferred from sick to still healthy (but susceptible) MRL/MpJ mice. Unpurified splenocytes and CD3⁺ T cells both efficiently induced AIP, while CD4⁺ and CD8⁺ T cells alone, as well as splenocytes from healthy mice, were insufficient to trigger the disease. Strikingly, CD4⁺CD44^high memory T cells, although transferred at lower numbers than other T cells, also induced AIP in recipient mice. Employing a modified experimental design, we also evaluated the effects of regulatory T cells (T_regs) on the progression of AIP in already diseased mice. Under the given experimental conditions, there was no significant suppressive effect of adoptively transferred T_regs on pancreatic histopathology. The results of our studies suggest a key role of T cell‐mediated processes in murine AIP. The effects of CD4⁺CD44^high memory T cells are in accordance with genetic studies of our group, which had previously implicated this cell type into the pathogenesis of AIP. In follow‐up studies, we will focus on the interplay of cellular and humoral autoimmunity in the context of AIP.     

Unique Properties of a Cytotoxic CD4+CD8+ Intraepithelial T-Cell Line Established from the Mouse Intestinal Epithelium

Growth factor-dependent gut intraepithelial lymphocyte (IEL) cell lines were established from a long-term in vitro culture of BALB/c IEL with syngeneic irradiated spleen cells in the presence of concanavalin A-stimulated spleen supernatant fluids. The cell lines were preferentially consisted of very limited thymoindependent subsets of IEL; i.e., Thy-1⁺ CD5^–TCRαβ⁺ CD4⁺CD8 α⁺β^– (double-positive; DP) IEL and Thy-1⁺ CD5^– TCRαβ⁺ CD4^–CD8α⁺β^– (CD8 single-positive; CD8 SP) IEL. The CD8 SP IEL cell line had cytotoxic activities and was triggered to proliferate by T-cell receptor (TCR)-directed stimuli. The DP IEL cell line expressed high levels of the CD3-TCRαβ, exhibited cytotoxic activity in redirected lysis assays, and had perforin in the cytoplasm, indicating the functional maturity of this cell line. However, the DP IEL cell line did not proliferate in response to TCRαβ-directed stimuli, which indicated that TCRαβ-mediated signalling was able to initiate cytotoxic function but not to induce proliferation of the DP IEL cell line. Although both cell lines were shown to have functional competence, they expressed J11d antigen which marks immaturity in thymocyte differentiation pathways. These results indicate that the established thymoindependent DP and CD8 SP IEL cell lines have unique properties distinct from DP thymocytes and CD8 SP peripheral T cells. Together with a recent report on freshly isolated DP IEL (10), the unique properties of the DP IEL cell line seems to support the notion that DP IEL may undergo a unique maturation process in the gut microenvironment.     

Retrovirus-mediated gene transfer into CD4+ and CD8+ human T cell subsets derived from tumor-infiltrating lymphocytes and peripheral blood mononuclear cells

Summary Studies were undertaken to test the susceptibility of individual T cell subpopulations to retroviral-mediated gene transduction. Gene transfer into human tumor-infiltrating lymphocytes (TIL) or peripheral blood mononuclear cells (PBMC) was carried out by transduction with an amphotropic murine retroviral vector (LNL6 or N2) containing the bacterialneo ^R gene. The presence of theneo ^R gene in the TIL population was demonstrated by Southern blot analysis, detection of the enzymatic activity of the gene product and by the ability of transduced TIL to proliferate in high concentrations of G418, a neomycin analog that is toxic to eukaryotic cells. The presence of theneo ^R gene in TIL did not alter their proliferation or interleukin-2 dependence compared to nontransduced TIL. The differential susceptibility of CD4⁺ and CD8⁺ lymphoid cells to the retro-virus-mediated gene transfer was then tested. Transduction of heterogeneous TIL cultures containing both CD4⁺ and CD8⁺ cells resulted in gene insertion into both T cell subsets with no preferential transduction frequency into either CD4⁺ or CD8⁺ cells. In other experiments highly purified CD4⁺ and CD8⁺ T cell subpopulations from either TIL or PBMC could be successfully transduced with theneo ^R gene as demonstrated by Southern blot analysis and detection of the gene product neophosphotransferase activity. No such activity or vector DNA could be detected in controls of nontransduced cells. In these highly purified cell subsets the distinctive T cell phenotypic markers were continually expressed after transduction, G418 selection and long-term growth. Clinical trials have begun in patients with advanced cancer using heterogeneous populations of CD4⁺ and CD8⁺ gene-modified TIL. Current address: Bone Marrow Transplantation, Hadassah University Hospital, 91120 Jerusalem, Israel     

Heterogeneity of CD4 and CD8+ memory T cells in localized and generalized Wegener's granulomatosis

Memory T cells display phenotypic heterogeneity. Surface antigens previously regarded as exclusive markers of naive T cells, such as L-selectin (CD62L), can also be detected on some memory T cells. Moreover, a fraction of CD45RO⁺ (positive for the short human isoform of CD45) memory T cells reverts to the CD45RA⁺ (positive for the long human isoform of CD45) phenotype. We analyzed patients with biopsy-proven localized Wegener's granulomatosis (WG) (n = 5), generalized WG (n = 16) and age- and sex-matched healthy controls (n = 13) to further characterize memory T cells in WG. The cell-surface expression of CD45RO, CD45RA, CD62L, CCR3, CCR5 and CXCR3 was determined on blood-derived T cells by four-color flow cytometric analysis. The fractions of CCR5⁺ and CCR3⁺ cells within the CD4⁺CD45RO⁺ and CD8⁺CD45RO⁺ memory T cell populations were significantly expanded in localized and generalized WG. The mean percentage of Th1-type CCR5 expression was higher in localized WG. Upregulated CCR5 and CCR3 expression could also be detected on a fraction of CD45RA⁺ T cells. CD62L expression was seen on approximately half of the memory T cell populations expressing chemokine receptors. This study demonstrates for the first time that expression of the inducible inflammatory chemokine receptors CCR5 and CCR3 on CD45RO⁺ memory T cells, as well as on CD45RA⁺ T cells ('revertants'), contributes to phenotypic heterogeneity in an autoimmune disease, namely WG. Upregulated CCR5 and CCR3 expression suggests that the cells belong to the effector memory T cell population. CCR5 and CCR3 expression on CD4⁺ and CD8⁺ memory T cells indicates a potential to respond to chemotactic gradients and might be important in T cell migration contributing to granuloma formation and vasculitis in WG.     

Characteristics of circulating CD31+ cells from patients with coronary artery disease

Recently, we reported the properties of CD31‐expressing cells in healthy individuals. However, the characteristics of CD31‐expressing cells derived from coronary artery disease (CAD) patients remain unknown. This study aimed to investigate the relationship between circulating CD31⁺ cells and CAD as well as their biological characteristics. Analysis with flow cytometry revealed that CD31⁺ cells (C‐CD31) from the peripheral blood (PB) of CAD patients exhibited low levels of T‐cell marker and high levels of macrophage marker compared with the PB‐CD31⁺ cells from healthy individuals (H‐CD31). In addition, the expression levels of multiple pro‐angiogenic and chemokine genes were significantly down‐regulated in C‐CD31. However, inflammatory gene IL‐1α was highly up‐regulated in C‐CD31. Patients with unstable angina (UA) had significantly more CD31⁺ cells in the PB than healthy control group (P < 0.001). Moreover, there were significant correlations between the number of CD31⁺ cells and cardiovascular (CV) disease activity (R = 0.318, P = 0.006) and the number of diseased coronaries (R = 0.312, P = 0.005). For the diagnostic category of UA, the area under curve was 0.803 (P < 0.001). In conclusion, C‐CD31 have impaired angiogenic potential and the number of circulating CD31⁺ cells were correlated with CV risk. These findings may contribute to the understanding of the pathogenesis of CAD.     

Metallothionein in human immunomagnetically selected CD34+ haematopoietic progenitor cells

Human umbilical CD34⁺ immature haematopoietic cells were rapidly and efficiently obtained from light density MNC (mononuclear cells) by MACS (magnetic cell sorting). An ex vivo expanded population of CD34⁺ was cultured in serum‐free medium supplemented with cytokines FL (flt3 ligand), SCF (stem cell factor) and TPO (thrombopoietin) in order to obtain a sufficient number of CD34⁺ cells. CD34⁺ cells expanded from cord blood for 7 days were demonstrated to increase in the absolute number of CD34⁺ cells by 5.12±2.47‐fold (mean±S.D., n=3). Flow cytometric analysis demonstrated that the percentage of CD34 antigen expression after expansion of the culture was 97.81±1.07%, whereas it was 69.39±10.37% in none‐expanded CD34⁺ cells (mean±S.D., n=3), thus defining a system that allowed extensive amplification accompanied by no maturation. MTs (metallothioneins), low molecular weight, cysteine‐rich metal‐binding proteins, exhibit various functions, including metal detoxification and homoeostasis. We here examined the expression pattern of functional members of the MT gene family in immature CD34⁺ cells and compared it with more mature CD34^? cells in order to strengthen the proposed function of MT in differentiation. Cells were cultured in RPMI 1640 medium, with or without different zinc supplements for 24 h. Relative quantitative expression of MT isogenes in the mature CD34^? cells was higher than in the immature CD34⁺ cells. IHC (immunohistochemical staining) revealed an increased MT protein biosynthesis in CD34^? cells, greater than in CD34⁺ cells. Therefore, the role of MT in differentiation of human haematopoietic progenitor cells from human cord blood is reported for the first time.     

CD45+/CD133+ positive cells expanded from umbilical cord blood expressing PDX‐1 and markers of pluripotency

UCB (human umbilical cord blood) contains cells able to differentiate into non‐haematopoietic cell lineages. It also contains cells similar to primitive ESCs (embryonic stem cells) that can differentiate into pancreatic‐like cells. However, few data have been reported regarding the possibility of expanding these cells or the differential gene expression occurring in vitro. In this study, we expanded formerly frozen UCB cells by treatment with SCF (stem cell factor) and GM‐CSF (granulocyte–macrophage colony stimulating factor) in the presence of VPA (valproic acid). Gene expression profiles for beta cell differentiation and pluripotency (embryo stem cell phenotype) were analysed by RT‐PCR and immunocytochemistry. The results show a dramatic expansion (>150‐fold) of haematopoietic progenitors (CD45⁺/CD133⁺) which also expressed embryo markers of pluripotency (nanog, kfl‐4, sox‐2, oct‐3/4 andc‐myc), nestin, and pancreatic markers such as pax‐4, ngn‐3, pdx‐1 and syt‐1 (that is regulated by pdx‐1 and provides the cells with a Ca⁺⁺ regulation mechanism essential for insulin exocytosis). Our results show that UCB cells can be expanded to produce large numbers of cells of haematopoietic lineage that naturally (without the need of retroviral vectors or transposons) express a gene pattern compatible with endocrine pancreatic precursors and markers of pluripotency. Further investigations are necessary to clarify, first, whether in this context, the embryogenes expressed are functional or not, and secondly, since these cells are safer than cells transfected with retroviral vectors or transposons, whether they would represent a potential tool for clinical application.     

Increased CD11b+ Gr‐1+ cell population in the placenta after infection with Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular protozoan pathogen that can cross the placenta, resulting in congenital toxoplasmosis with severe fetal brain abnormalities. The molecular mechanisms of immune responses against T. gondii infection in the placenta have largely remained unclear. An analytical method for characterizing phenotypes of immune cells in the placenta by flow cytometry was established and it was found that numbers of CD11b⁺ Gr‐1⁺ cells in the placenta increased significantly after T. gondii infection. These results suggest that innate immune responses play an important role in immunity against T. gondii infection via the feto‐maternal interface.     

Decreased regulatory T‐cells and CD4+/CD8+ ratio correlate with disease onset and progression in patients with generalized vitiligo

The aim of present study was to evaluate CD4⁺/CD8⁺ ratio and CD4⁺CD25^hiFoxP3⁺ Tregs in GV patients with reference to their effect on disease onset and progression. Flow cytometry was used for determination of CD4⁺/CD8⁺ ratio and Tregs in 82 patients and 50 controls. CD8⁺ T‐cell counts were significantly higher in GV patients as compared with controls (p = 0.003). Active GV patients showed higher CD8⁺ T‐cell counts compared with stable GV patients (p = 0.001). The CD4⁺/CD8⁺ ratio decreased significantly in patients as compared with controls (p = 0.001). Moreover, the ratio in active GV patients significantly lowered as compared with stable GV patients (p = 0.002). Significant decrease in Treg cell percentage and counts in GV patients was observed compared with controls (p = 0.009, p = 0.008) with significant reduction in FoxP3 expression (p = 0.024). Treg cell percentage and counts were significantly decreased in active GV patients compared with stable GV patients (p = 0.007, p = 0.002). Our results suggest that an imbalance of CD4⁺/CD8⁺ ratio and natural Tregs in frequency and function might be involved in the T‐cell mediated pathogenesis of GV and its progression.     

Analysis of human V H gene repertoire expression in peripheral CD19+ B cells

Using CD19 B-cell selection and polymerase chain reaction-amplified cDNA libraries, we analyzed the peripheral immunoglobulin heavy chain variable repertoire of three healthy adult donors. Here we report that most of the CD19+ circulating B cells expressed unmutated V _H-D-J_H rearrangements. By specific V _H family hybridization, we show that V _H gene family utilization in the periphery roughly corresponds to the complexity of these families in the germline and appears to be relatively constant among the analyzed subjects. However, sequence data of clones picked at random from one IgM cDNA library reveals that in spite of this random utilization, the V _H gene expression in naive circulating B cells is highly biased towards the expression of a limited set of V _H genes. As previously reported by others, this restricted mechanism is also found for the D and J _H segments.The nucleotide sequence data reported in this paper have been submitted to the GenBank/EMBL nucleotide sequence database and have been assigned the accession numbers Z47213-Z47243 and Z47349     

Non-linear patterns in age-related DNA methylation may reflect CD4+ T cell differentiation

DNA methylation (DNAm) is an important epigenetic process involved in the regulation of gene expression. While many studies have identified thousands of loci associated with age, few have differentiated between linear and non-linear DNAm trends with age. Non-linear trends could indicate early- or late-life gene regulatory processes. Using data from the Illumina 450K array on 336 human peripheral blood samples, we identified 21 CpG sites that associated with age (P<1.03E-7) and exhibited changing rates of DNAm change with age (P<1.94E-6). For 2 of these CpG sites (cg07955995 and cg22285878), DNAm increased with age at an increasing rate, indicating that differential DNAm was greatest among elderly individuals. We observed significant replication for both CpG sites (P<5.0E-8) in a second set of peripheral blood samples. In 8 of 9 additional data sets comprising samples of monocytes, T cell subtypes, and brain tissue, we observed a pattern directionally consistent with DNAm increasing with age at an increasing rate, which was nominally significant in the 3 largest data sets (4.3E-15<P<0.039). cg07955995 and cg22285878 reside in the promoter region of KLF14, which encodes a protein involved in immune cell differentiation via the repression of FOXP3. These findings may suggest a possible role for cg07955995 and cg22285878 in immunosenescence.     

Phenotypic characteristics of aged CD4+ CD28null T lymphocytes are determined by changes in the whole‐genome DNA methylation pattern

Aging is associated with a progressive loss of the CD28 costimulatory molecule in CD4⁺ lymphocytes (CD28^null T cells), which is accompanied by the acquisition of new biological and functional properties that give rise to an impaired immune response. The regulatory mechanisms that govern the appearance and function of this cell subset during aging and in several associated inflammatory disorders remain controversial. Here, we present the whole‐genome DNA methylation and gene expression profiles of CD28^null T cells and its CD28⁺ counterpart. A comparative analysis revealed that 296 genes are differentially methylated between the two cell subsets. A total of 160 genes associated with cytotoxicity (e.g. GRZB, TYROBP, and RUNX3) and cytokine/chemokine signaling (e.g. CX3CR1, CD27, and IL‐1R) are demethylated in CD28^null T cells, while 136 de novo‐methylated genes matched defects in the TCR signaling pathway (e.g. ITK, TXK, CD3G, and LCK). TCR‐landscape analysis confirmed that CD28^null T cells have an oligo/monoclonal expansion over the polyclonal background of CD28⁺ T cells, but feature a Vβ family repertoire specific to each individual. We reported that CD28^null T cells show a preactivation state characterized by a higher level of expression of inflammasome‐related genes that leads to the release of IL‐1β when activated. Overall, our results demonstrate that CD28^null T cells have a unique DNA methylation landscape, which is associated with differences in gene expression, contributing to the functionality of these cells. Understanding these epigenetic regulatory mechanisms could suggest novel therapeutic strategies to prevent the accumulation and activation of these cells during aging.     

Enrichment of Murine CD68+CCR2+ and CD68+CD206+ Lung Macrophages in Acute Pancreatitis-Associated Acute Lung Injury

Acute lung injury (ALI) is an important cause of mortality in critically ill patients. Acute pancreatitis (AP) is one of the risk factors for developing this syndrome. Among the inflammatory cells, macrophages have a key role in determining the severity of the acute lung injury. In the lungs, macrophages constitute a heterogeneous cell population distributed in different compartments. Changes in not only the macrophage count, but also in their phenotype have been seen during the course of lung injury. A murine ductal ligation model of acute pancreatitis showed substantial morphological changes in the pancreas and lungs. Immunohistochemistry showed neutrophil recruitment into both organs after 9 hours and later on. F4/80⁺ cells in the pancreas increased in the ligated animals, though there was not a significant difference in their number in the lungs as compared to sham operated animals. Flow cytometry analysis of lung macrophages demonstrated an enrichment of F4/80⁻ CD68⁺CCR2⁺ and F4/80⁻ CD68⁺CD206⁺ lung macrophages in ligated animals (AP) as compared to the sham operated group. The level of interleukin-6 in plasma increased 3 hours after ligation compared to the sham operated group, as a first indicator of a systemic inflammatory response.This study suggests a role for F4/80⁻ CD68⁺ macrophages in the pathogenesis of acute lung injury in acute pancreatitis. Studying lung macrophages for different phenotypic markers, their polarization, activation and recruitment, in the context of acute lung injury, is a novel area to potentially identify interventions which may improve the outcome of acute lung injury.