TLR2‐dependent amelioration of allergic airway inflammation by parasitic nematode type II MIF in mice

In our previous studies, the recombinant type II macrophage migration inhibitory factor homologue (rAs‐MIF) secreted from Anisakis simplex suppressed experimental inflammation mouse model through IL‐10 production and CD4⁺CD25⁺Foxp3⁺ T‐cell recruitment. Also, TLR2 gene expression was significantly increased following rAs‐MIF treatment. To know the relation between TLR2 and amelioration mechanisms of rAs‐MIF, we induced allergic airway inflammation by ovalbumin and alum with or without rAs‐MIF under TLR2 blocking systems [anti‐TLR2‐specific antibody (α‐mTLR2 Ab) treatment and using TLR2 knockout mice]. As a result, the amelioration effects of rAs‐MIF in allergic airway inflammation model (diminished inflammation and Th2 response in the lung, increased IL‐10 secretion, CD4⁺CD25⁺Foxp3⁺ T‐cell recruitment) were diminished under two of the TLR2 blocking model. The expression of TLR2 on the surface of lung epithelial cell was significantly elevated by rAs‐MIF treatment or Pam3CSK (TLR2‐specific agonist) treatment, but they might have some competition effect on the elevation of TLR2 expression. In addition, the elevation of IL‐10 gene expression by rAs‐MIF treatment was significantly inhibited by α‐mTLR2 Ab or Pam3CSK pretreatment. In conclusion, anti‐inflammatory effects of the rAs‐MIF on OVA‐induced allergic airway inflammation might be closely related to TLR2.     

Heligmosomoides bakeri antigen rescues CD4-positive T cells from glucocorticoid-induced apoptosis by Bcl-2 protein expression

Heligmosomoides bakeri infection in mice is associated with a dominant CD4⁺ T‐cell response and with the activity of natural Treg cells with CD4⁺CD25⁺ phenotype. The polarization of Th2 T‐cell phenotype and the increase in the CD4⁺CD25⁺ T cell population are regulated by glucocorticoids that induce apoptosis in CD4⁺CD25^? T cells and inhibit apoptosis in CD4⁺CD25⁺ T cells. However, exposure of mice to H. bakeri antigen induces a high glucocorticoid concentration in serum and a reduction in the number of CD4‐positive; CD4⁺CD25^? and CD4⁺CD25⁺ apoptotic T cells in mesenteric lymph node cells. In this study to evaluate the in vitro effect of the anti‐apoptotic property of H. bakeri antigen on T cells, apoptosis of these cells was induced by glucocorticoids‐dexamethasone (Dex). Excretory–secretory (ES) antigen of the nematode prevented Dex‐induced apoptosis in CD4‐positive T cells with CD4⁺CD25^? and CD4⁺CD25^High phenotype by Bcl‐2 protein expression. Contrary to the effect on CD4‐positive T cells, survival of CD8⁺ T cells was not connected with expression of Bcl‐2 protein. This suggest that H. bakeri antigen modulates CD4‐positive T cell sensitivity to glucocorticoid‐induced apoptosis by induction of Bcl‐2 protein.     

Differential virus‐specific CD8+ T‐cell epitope repertoire in hepatitis C virus genotype 1 versus 4

Virus‐specific CD8⁺ T‐cell responses play an important role in the outcome of hepatitis C virus (HCV) infection. To date, most HCV‐specific CD8⁺ T‐cell epitopes have been defined in HCV genotype 1 infection. In contrast, the HCV genotype 4‐specific CD8⁺ T‐cell response is poorly defined. Here, we analysed whether known HCV‐specific CD8⁺ T‐cell epitopes are also recognized in HCV genotype 4‐infected patients and set out to identify the first HCV genotype 4‐specific CD8⁺ T‐cell epitopes. We studied patients chronically infected with HCV genotype 1 (n = 20) or 4 (n = 21) using 91 well‐described HCV‐specific epitope peptides. In addition, we analysed 24 genotype 4‐infected patients using 40 epitope candidates predicted using an in silico approach. HCV‐specific CD8⁺ T‐cell responses targeting previously described epitopes were detectable in the majority of genotype 1‐infected patients (11 of 20). In contrast, patients infected with HCV genotype 4 rarely targeted these epitopes (4 of 21; P = .0247). Importantly, we were able to identify eight novel HCV genotype 4‐specific CD8⁺ T‐cell epitopes. Only one of these epitopes was shared between genotype 1 and genotype 4. These results indicate that there is little overlap between CD8⁺ T‐cell repertoires targeting HCV genotype 1 and 4. Prophylactic vaccination studies based on HCV genotype 1 are currently underway. However, in countries with the highest prevalence of HCV infection, such as Egypt, most patients are infected with HCV genotype 4. Thus, prophylactic vaccination strategies need to be adapted to HCV genotype 4 before their application to regions where HCV genotype 4 is endemic.     

Immunophenotypic analysis of T‐acute lymphoblastic leukemia. A CD5‐based ETP‐ALL perspective of non‐ETP T‐ALL

T‐cell antigens [CD5,CD1a,CD8] define early T‐cell precursor acute lymphoblastic leukemia (ETP‐ALL). To understand immature T‐ALL of which ETP‐ALL is part, we used these antigens to subcategorize non‐ETP T‐ALL for examining expression of myeloid/stem cell antigens (M/S) and clinical features. Using CD5 (+/?) to start categorization, we studied 69 routinely immunophenotyped patients with T‐ALL. CD5^? was a homogenous (CD8,CD1a)^? M/S⁺ ETP‐ALL group (n = 9). CD5⁺ cases were (CD8,CD1a)^? pre‐T‐ALL (n = 22) or (CD8,CD1a)⁺ (n = 38) thymic/cortical T‐ALL; M/S⁺ 20/22 (90.91%) in former and 22/38 (57.89%) in latter (P = 0.007). ETP‐ and pre‐T‐ALL together (CD1a^?,CD5^?/+ immature T‐ALL group) were nearly always M/S⁺ (29/31; 93.55%). In multivariate analysis, only ETP‐ALL predicted poor overall survival (P = 0.02). We conclude (i) CD5 negativity in T‐ALL almost always means ETP‐ALL. CD1a and CD8 negativity, as much as CD5, marks immaturity in T‐ALL, and the CD5^+/?/CD1a^?/CD8^? immature T‐ALL group needs further study to understand the biology of the T‐ALL–myeloid interface. (ii) ETP‐ALL patients may be pre‐T‐ALL if CD2⁺; CD2⁺, conversely, CD5^?/CD1a^?/CD8^? pre‐T ALL patients are ETP‐ALL. (iii) Immunophenotypic workup of T‐ALL must not omit CD1a, CD5, CD8 and CD2, and positivity of antigens should preferably be defined as recommended for ETP‐ALL, so that this entity can be better evaluated in future studies of immature T‐ALL, a group to which ETP‐ALL belongs. (iv) ETP‐ALL has poor prognosis.     

Increased Fas ligand expression of peripheral B‐1 cells correlated with CD4+ T‐cell apoptosis in filarial‐infected patients

Cellular hyporesponsiveness observed during helminth infections is attributed to factors such as antigen‐presenting cells (APC) dysfunction, increased interleukin‐10(IL‐10), regulatory T cells and induction of CD4⁺ T (Th)‐cell apoptosis. Increased Fas ligand (FasL) expression on the surface of B‐1 cells and induction of apoptosis of Th cells by FasL‐expressing B‐1 cells due to helminth infection were demonstrated in murine model of helminth infection where as profile of FasL expression, Th‐cell apoptosis and correlation between these two populations of cells in clinical filariasis remain unknown. In this study, we have scored the profile of apoptotic Th‐cell population and FasL‐expressing B‐1 cells in different clinical categories of filariasis. The peripheral apoptotic T‐helper cells were significantly increased in filarial patients compared to endemic controls. Expression of FasL on the surface of peripheral B‐1 cells increased in filarial patients and positively correlated with peripheral apoptotic T‐helper cells indicating FasL‐expressing B‐1 cells may be one of the important mediators of Th‐cell apoptosis and immune anergy during filarial pathology.     

A prospective study of T‐ and B‐lymphocyte subpopulations,CD81 expression levels on B cells and regulatory CD4+CD25+CD127low/−FoxP3+ T cells in patients with chronic HCV infection during pegylated interferon‐alpha2a plus ribavirin treatment

Summary. Resolution of hepatitis C virus (HCV) infection requires a complex interplay between innate and adaptative immune responses. The role of lymphocyte subpopulations during combined antiviral treatment remains to be defined. This study was conducted to assess the effect of pegylated interferon‐alpha2a (pegIFN‐α2a) and ribavirin treatment on peripheral blood lymphocytes, mainly on CD81 expression on B cells and CD4⁺CD25⁺CD127^low/?FoxP3⁺ regulatory T cells (Tregs) in patients with chronic HCV infection. Thirty‐five patients with chronic HCV infection who started pegIFN‐α2a and ribavirin treatment were enrolled. Peripheral blood mononuclear cells (PBMC) were obtained at baseline before treatment (BT), mid‐treatment (MT), the end of treatment (ET) and 24 weeks post‐treatment (PT). During combined antiviral treatment, a significant decrease in the percentage of CD3⁺, CD8⁺, CD3⁺gamma/delta (γδ)⁺, CD19⁺ lymphocyte subpopulations and Tregs was observed. There was also a significant increase in the percentage of the CD4⁺ lymphocyte subpopulation and in CD81 expression levels on CD19⁺ B cells when BT was compared with ET (all P < 0.05). Seventeen patients were nonresponders (NR) and 18 had a sustained virological response (SVR). At baseline, NR patients had higher CD81 expression levels on CD19⁺ B cells (P = 0.017) and a higher Tregs percentage (P = 0.025) than SVR patients. Our results suggest that immunomodulation fluctuates during antiviral treatment and that percentage CD81 expression levels on B cells and Tregs might be useful as an immunological prognostic factor for pegIFN‐α2a and ribavirin treatment response in chronic HCV infection.     

Role of F4/80+Mac‐1high adherent non‐parenchymal liver cells in concanavalin A‐induced hepatic injury in mice

Aim: Non‐parenchymal liver cells (NPLC) play an important role in the regulation of immune responses and the inflammatory process. In this study, we hypothesized that F4/80⁺Mac‐1^high+ cells were involved in the regulative feedback‐modulated regulation of inflammatory responses during concanavalin A (Con A)‐induced hepatitis. Methods: Hepatitis was induced in BALB/c mice by the intravenous injection of Con A. Liver injury was assessed using serum aminotransferase and pathology. The function of NPLC was assessed by FACS analysis. Accessory cell function of adherent Con A NPLC was performed with an ovalbumin specific T‐helper 1 (Th1) clone proliferation assay. The culture supernatant nitric oxide (NO) content was quantified by the Griess reaction. Inducible NO synthase (iNOS) expression was demonstrated by immunohistochemistry and Western blot analysis. Results: The number of hepatic F4/80⁺Mac‐1^high+cells increased in a time‐dependent manner after Con A administration, which consequently suppressed Th1 cell proliferation by a mechanism likely to involve NO. The iNOS expression of NPLC was elevated at 24 h post‐Con A injection. In nude mice, F4/80⁺Mac‐1^high+cells did not increase in the Con A‐treated liver; the NPLC did not suppress Th1 clone proliferation. Conclusion: These findings suggest that the in vivo activation of F4/80⁺Mac‐1^high+cells by Con A administration suppresses Th1 cell proliferation by increasing NO, and subsequently reducing liver injury.     

The immunosuppressive effects of CD4+CD25+ regulatory T cells on dendritic cells in patients with chronic hepatitis B

The characteristics and functions of CD4⁺CD25⁺ regulatory T cells (Tregs) have been well defined in murine and human systems. However, the interaction or crosstalk between CD4⁺CD25⁺ Tregs and dendritic cells (DCs) remains controversial. In this study, the effects of chronic hepatitis B (CHB) CD4⁺CD25⁺ Tregs on the maturation and function of monocyte‐derived DCs were examined. The results showed that CD4⁺CD25⁺ render the DCs inefficient as antigen‐presenting cells (APCs) despite prestimulation with CD40 ligand. This effect was marginally reverted by applying neutralizing antibodies (Abs) to IL‐10 and TGF‐β. There were an increased IL‐10 and TGF‐β secretion and reduced expression of costimulatory molecules in DC. Thus, in addition to a direct suppressor effect on CD4⁺T cells, CD4⁺CD25⁺ may modulate the immune response through DCs in CHB patients.     

Idiotype-specific T lymphocytes in monoclonal gammopathies: evidence for the presence of CD4+ and CD8+ subsets

Tumour-specific CD4⁺ T helper (Th) and CD8⁺ T cytotoxic (Tc) cells may participate in the control and eradication of tumour cells. In the present study, idiotype-specific stimulation of CD4⁺ and CD8⁺ blood T cells from patients with monoclonal gammopathy of undetermined significance and patients with untreated multiple myeloma stage I was examined. Activation was measured in the CD4⁺ and CD8⁺ subsets enriched by magnetic microbeads as the incorporation of ³H-thymidine and the secretion of interferon (IFN)-γ, interleukin (IL)-2 and IL-4 by single cells using the enzyme-linked immunospot assay. Idiotype-specific T cells were found in four of seven patients. Stimulation was mainly confined to the CD4⁺ subset in three of the four responding patients. This type of response was major histocompatibility complex (MHC) class II restricted as it could be inhibited by monoclonal antibodies against MHC class II (HLA-DR), but not against class I (HLA-ABC) molecules. Idiotype-specific CD8⁺ T cells were also demonstrated in these patients although at a lower frequency. One patient showed a strong and dominating activation of CD8⁺ T cells which could be blocked by antibodies against HLA-ABC but not against HLA-DR. Idiotype-specific CD4⁺ or CD8⁺ T cells were mainly of the type-1 subsets as judged by their secretion of IFN-γ and IL-2. Thus, this study provides evidence for the presence of idiotype-specific and MHC-restricted CD4⁺ and CD8⁺ T cells of the type-1 subsets in patients with monoclonal gammopathies. Such T cells with the potential to control the growth of tumour B cells may be a suitable target for immunotherapeutic interventions in patients.     

Frequency of specific CD8+ T cells for a promiscuous epitope derived from Trypanosoma cruzi KMP‐11 protein in chagasic patients

The K1 peptide is a CD8 ⁺ T cell HLA‐A*0201‐restricted epitope derived from the Trypanosoma cruzi KMP‐11 protein. We have previously shown that this peptide induces IFN‐γ secretion by CD8⁺T cells. The aim of this study was to characterize the frequency of K1‐specific CD8⁺T cells in chagasic patients. Nineteen HLA‐A2⁺individuals were selected from 50 T. cruzi infected patients using flow cytometry and SSP‐PCR assays. Twelve HLA‐A*0201⁺noninfected donors were included as controls. Peripheral blood mononuclear cells were stained with HLA‐A2‐K1 tetramer, showing that 15 of 19 infected patients have K1‐specific CD8⁺T cells (0·09–0·34% frequency) without differences in disease stages or severity. Of note, five of these responders were A*0205, A*0222, A*0226, A*0259 and A*0287 after molecular typing. Thus, a phenotypic and functional comparison of K1‐specific CD8⁺T cells from non‐HLA‐A*0201 and HLA‐A*0201⁺infected patients was performed. The results showed that both non‐HLA‐A*0201 and HLA‐A*0201⁺individuals have a predominant effector memory CD8⁺T cell phenotype (CCR7?, CD62L?). Moreover, CD8⁺T cells from non‐HLA‐A*0201 and HLA‐A*0201⁺individuals expressed IL‐2, IFN‐γ and perforin without any differences. These findings support that K1 peptide is a promiscuous epitope presented by HLA‐A2 supertype molecules and is highly recognized by chagasic patients.     

Host Th1/Th2 immune response to Taenia solium cyst antigens in relation to cyst burden of neurocysticercosis

Neurocysticercosis (NCC), Taenia solium larval infection of the brain, is an important cause of acquired seizures in endemic countries, which relate to number, location and degenerating cysts in the brain. Multicyst infections are common in endemic countries although single‐cyst infection prevails in India. Single‐cyst infections in an endemic country suggest a role for host immunity limiting the infection. This study examined ex vivo CD4⁺ T cells and in vitro Th1 and Th2 cytokine responses to T. solium cyst antigens of peripheral blood mononuclear cells of healthy subjects from endemic and nonendemic regions and of single‐ and multicyst‐infected patients for association with cyst burden of NCC. T. solium cyst antigens elicited a Th1 cytokine response in healthy subjects of T. solium‐endemic and T. solium‐non‐endemic regions and those with single‐cyst infections and a Th2 cytokine response from subjects with multicyst neurocysticercosis. Multicyst neurocysticercosis subjects also exhibited low levels of effector memory CD4⁺ T cells. Th1 cytokine response of T. solium exposure and low infectious loads may aid in limiting cyst number. Th2 cytokines and low effector T cells may enable multiple‐cyst infections to establish and persist.     

Persistent hepatitis C virus (HCV) infection impairs HCV‐specific cytotoxic T cell reactivity through Mcl‐1/Bim imbalance due to CD127 down‐regulation

Summary. In persistent hepatitis C virus (HCV) infection, HCV‐specific cytotoxic T lymphocyte (CTL) reactivity is impaired and this affects HCV control. Interleukin‐7 receptor (CD127) expression on these cells could regulate CTL reactivity through Mcl‐1/Bim balance modulation. Bim is a pro‐apoptotic molecule blocked by the action of Mcl‐1. Mcl‐1/Bim expression and T cell reactivity on HCV‐specific CTLs were compared according to CD127 phenotype. Peripheral blood lymphocytes (PBL) from HLA‐A2⁺ HCV⁺ patients were obtained. HCV‐specific CTLs were visualized by staining PBL with anti‐CD8 and HLA‐A2/peptide pentameric complexes (pentamer). Mcl‐1/Bim/CD127 phenotype of HCV‐specific CTLs was tested by staining detectable CD8⁺/pentamer⁺ cells with anti‐Mcl‐1/Bim/CD127 antibodies. HCV‐specific CTL proliferation ability after specific in vitro challenge was tested in the presence and absence of pancaspase inhibitor z‐VAD‐fmk. All stained cells were analysed by flow cytometry. CD127^low‐expressing HCV‐specific CTLs associated with high HCV viraemia, while CD127^high correlated with undetectable viral loads (P < 0.001). Directly ex vivo, pentamer⁺ cell frequency was similar according to CD127 expression level. Nevertheless, CD127^low pentamer⁺ cell proliferation after specific in vitro challenge was impaired (P < 0.05), although this was corrected by z‐VAD‐fmk treatment (P < 0.05). Mcl‐1 expression was low directly ex vivo (P < 0.01), and Bim was up‐regulated after antigen encounter (P < 0.05) of CD127^low pentamer⁺ cells. The ex vivo difference between Mcl‐1 and Bim expression on pentamer⁺ cells correlated positively with CD127 expression level (P < 0.001) and with pentamer⁺ cell reactivity (P < 0.05). In summary, a low ex vivo Mcl‐1 expression and Bim up‐regulation after antigen encounter are involved in CD127^low HCV‐specific CTL hyporeactivity during chronic infection, but it can be overcome by apoptosis blockade.     

Prevention of acute graft‐versus‐host disease by humanized anti‐CD26 monoclonal antibody

CD26 (DPP4) is a T cell costimulatory molecule as well as T cell activation marker, and CD26⁺ T cells are accumulated in inflamed tissues, such as rheumatoid synovitis and autoimmune thyroiditis. In the present study, we found accumulation of CD26⁺ T cells in graft‐versus‐host disease (GVHD) target organs. To expand our in vitro findings to an in vivo system, we examined CD26‐dependent organ injury in a xenogeneic GVHD (x‐GVHD) murine model. Following intraperitoneal injection of human peripheral blood mononuclear cells into non‐obese diabetic severe combined immunodeficiency/γ_c^?/? mice (hu‐PBL‐NOG mice), the mice exhibited the onset of GVHD symptoms associated with the presence of CD26^high human lymphocytes in the peripheral blood and GVHD target tissues. Administration of humanized anti‐human CD26 monoclonal antibody (mAb) decreased x‐GVHD severity and prolonged survival in hu‐PBL‐NOG mice without loss of engraftment of human T cells, while increasing doses of CTLA4‐ immunoglobulin fusion protein diminished engraftment of human lymphocytes. Importantly, anti‐CD26 mAb treatment preserved the graft‐versus‐leukaemia effects in studies using cotransplantation of P815 murine leukaemic cells. In addition, CD26⁺ lymphocytes infiltrated the GVHD patients' target tissues. Altogether, our data indicate a role for CD26 in the regulation of GVHD and point to CD26 as a novel target for therapeutic intervention in this disease.     

The effect of Echinococcus granulosus on spleen cells and TGF‐β expression in the peripheral blood of BALB/c mice

Cystic echinococcosis (CE) caused by the cestode Echinococcus granulosus (E. granulosus) is a zoonotic parasitic disease. The effective immune evasion mechanisms of E. granulosus allow it to parasitize its hosts. However, the status of the innate and adaptive immune cells and their contributions to E. granulosus progression remain poorly understood. In this study, we aimed to determine the impact of E. granulosus infection on T cells, NK cell responses and TGF‐β expression during the early infection phase in BALB/c mice. In E. granulosus infections, there was an increasing tendency in the percentage of CD4⁺CD25⁺ T cells and CD4⁺Foxp3⁺ T cells and peripheral blood TGF‐β levels and relative expression of the Foxp3 gene. Moreover, there were a decreasing tendency in the percentage of NK cells and NK cell cytotoxicity and the expression of NKG2D on NK cells. The TGF‐β1/Smad pathway was activated by E. granulosus in mice. Above results can be reversed by the inhibitor SB‐525334 (potent activin receptor‐like kinase 5 inhibitor). These results suggest that the TGF‐β/Smad pathway plays an important role in changes of T‐cell or NK cell responses. These results may contribute to revealing the preliminary molecular mechanisms in establishing hydatid infection.     

Enhanced high‐mobility group box 1 (HMGB1) modulates regulatory T cells (Treg)/T helper 17 (Th17) balance via toll‐like receptor (TLR)‐4‐interleukin (IL)‐6 pathway in patients with chronic hepatitis B

High‐mobility group box 1 (HMGB1) proteins are substantially up‐regulated in acute and chronic hepatitis. However, the immunopathogenic role of HMGB1 in patients with chronic hepatitis B (CHB) has not been elucidated. In this study, using a cohort of 36 CHB patients, we demonstrated a crucial role for HMGB1 to modulate balance between regulatory T (Treg) and T helper 17 (Th17) cells via the toll‐like receptor (TLR)‐4‐interleukin (IL)‐6 pathway. Serum HMGB1 levels were dramatically higher in CHB patients and increased along with liver injury, inflammation and fibrosis. Notably, HMGB1 increased along with decreased Treg/Th17 cells ratios in the periphery or intrahepatic microenvironment, which provides a clue for HMGB1 to favour Th17 responses whereas inhibit Treg responses. For in vitro studies, serum pools were constructed with serum from CHB patients at an advanced stage, whereas peripheral blood mononuclear cells (PBMC) pools were constructed with cells from those at an early stage. CHB‐serum significantly enhanced retinoic acid‐related orphan receptor‐γt (RORγt), whereas they inhibited forkhead box P3 (Foxp3) expression in CHB‐PBMC, which could be reversed by blocking of HMGB1, TLR4, or IL‐6. Besides, recombinant HMGB1 (rHMGB1) dose‐dependently up‐regulated RORγt whereas down‐regulated Foxp3 expression in CHB‐PBMC, and meanwhile, rHMGB1 enhanced TLR4 and IL‐6 expression in CHB‐PBMC. Moreover, the axis of HMGB1–TLR4‐IL‐6–Treg/Th17 required noncontact interactions between CD4 and non‐CD4 cells. In addition, rHMGB1 down‐regulated anti‐inflammatory proteins on CD4⁺CD25⁺ cells whereas up‐regulated pro‐inflammatory cytokines in CD4⁺CD25⁻ cells. In summary, enriched HMGB1 in CHB patients shifts Treg/Th17 balance to Th17 dominance via the TLR4‐IL‐6 pathway, which exacerbates liver injury and inflammation.     

Control of type 1 diabetes by CD4+Foxp3+ regulatory T cells: lessons from mouse models and implications for human disease

In recent years, there has been a revival of the concept of CD4⁺ regulatory T (T_reg) cells as being a central control point in various immune responses, including autoimmune responses and immunity to transplants, allergens, tumours and infectious microbes. The current literature suggests that T_reg cells are diverse in their phenotype and mechanism(s) of action, and as such, may constitute a myriad of naturally occurring and induced T cell precursors with variable degrees of regulatory potential. In this review, we summarize research from various laboratories, including our own, showing that CD4⁺Foxp3⁺ T_reg cells are critical in the control of type 1 diabetes (T1D) in mouse models and humans. In this review, we also discuss cellular and molecular determinants that impact CD4⁺Foxp3⁺ T_reg cell development and function and consequential resistance to organ‐specific autoimmune disease. Recent advances in the use of CD4⁺Foxp3⁺ T_reg cellular therapy to promote immunological tolerance in the absence of long‐term generalized immunosuppression are also presented. Copyright © 2009 John Wiley & Sons, Ltd.     

Differential in vitro CD4+/CD8+ T‐cell response to live vs. killed Leishmania major

Clinical trials of killed Leishmania vaccines showed a limited efficacy compared with leishmanization (LZ). The reason for this difference in protection against cutaneous leishmaniasis (CL) is not known and in vivo studies on T‐cell function may provide valuable information. Nevertheless, there are limited studies on the nature of the stimulatory effects of live vs. killed parasites on human T cells in vitro. A total of nine Leishmanin Skin Test+ volunteers with a history of self‐healing CL (HCL) and seven healthy volunteers were included in this study. 5,6‐carboxyfluroescein diacetate succinimidyl ester‐labelled CD4⁺/CD8⁺ lymphocytes were cultured with killed Leishmania Lysate (Killed LL) or live Leishmania major (Live LM) and analysed for proliferation using flow cytometry. Culture supernatants were used for cytokine titration. In HCL volunteers, upon stimulation with killed LL, the number of proliferated CD4⁺/CD8⁺ cells was significantly more than that of unstimulated (P < 0·001) or live LM stimulated (P < 0·05) cells, or cells from controls (CD4⁺/CD8⁺: P < 0·05/P < 0·001). Stimulation of CD4⁺ cells with Live LM (P < 0·001) or Killed LL (P < 0·05) induced a significantly higher IFN‐γ production compared with that of controls, but Live LM induced significantly (P < 0·05) more IFN‐γ than Killed LL. A significantly (P < 0·05) higher IFN‐γ production was observed when CD8⁺ cells were stimulated with Live LM. Cells from HCL volunteers showed significantly more IL‐10 production to Live LM stimulation compared with that of controls (CD4⁺: P < 0·05 /CD8⁺: P < 0·001) or cells stimulated with Killed LL (CD4⁺/CD8⁺: P < 0·001/P < 0·0005). Whereas Killed LL induced more proliferation response in purified T cells, Live LM induced cytokine production without significant induction of proliferation. The results from healed CL volunteers in this study could be implicated in further studies on T‐cell response in vaccinated individuals.     

Association between CD8+ T‐cell subsets and cardiovascular disease

Background

The findings of experimental studies suggest that the immune system plays a key role in atherosclerosis, but the clinical importance of different immune cells in cardiovascular disease remains poorly characterized. In this study we investigated the association between CD8⁺ T cells and carotid disease as well as development of cardiovascular disease events.

Methods

The study cohort comprised 700 subjects from the cardiovascular arm of the Malmö Diet and Cancer Study. Peripheral blood mononuclear cells, obtained at the 1991–1994 baseline investigation and stored at ?140 °C, were thawed and the different CD8⁺ T‐cell populations analysed by flow cytometry. Baseline carotid intima–media thickness and stenosis were assessed by ultrasonography and clinical events were monitored through validated national registers.

Results

Subjects with a high fraction of CD8⁺ T cells were characterized by decreased cytokine release from activated leucocytes, metabolic signs of insulin resistance and increased incidence of coronary events; hazard ratios (95% confidence intervals) for the second and third tertiles of CD8⁺ T cells were 2.57 (1.16, 5.67) and 2.61 (1.19, 5,71), respectively, in a Cox proportional hazards regression model. Correlations were found between the fraction of CD8⁺CD25⁺ T cells and the degree of carotid stenosis (r = 0.11, P < 0.01), and between the CD8⁺CD56^?IFN‐γ⁺ T‐cell fraction and the degree of stenosis (r = ?0.18, P < 0.005). The association between CD8⁺CD56^?IFN‐γ⁺ T cells and carotid stenosis remained significant after controlling for major cardiovascular disease risk factors.

Conclusion

This study provides prospective clinical evidence for a role of CD8⁺ T cells in cardiovascular disease and suggests the existence of CD8⁺ T‐cell subsets with different pathological functions.

     

Optimization of isolation and further characterization of umbilical cord blood‐derived very small embryonic/ epiblast‐like stem cells (VSELs)

Because of their small size and density, umbilical cord blood (UCB)‐derived very small embryonic/epiblast‐like stem cells (VSELs) are usually lost at various steps of UCB preparation. Accordingly, we noticed that a significant number of these cells, which are smaller than erythrocytes, are lost during gradient centrifugation over Ficoll‐Paque as well as during routine volume depletion of UCB units before freezing. To preserve these cells in final UCB preparations, we propose a relatively short and economical three‐step isolation protocol that allows recovery of approximately 60% of the initial number of Lin^?/CD45^?/CD133⁺ UCB‐VSELs present in freshly harvested UCB units. In this novel approach (i) UCB is lysed in a hypotonic ammonium chloride solution to deplete erythrocytes; (ii) CD133⁺ including VSELs cells are enriched by employing immunomagnetic beads; and subsequently (iii) Lin^?/CD45^?/CD133⁺ cells are sorted by fluorescence‐activated cell sorting. The whole isolation procedure takes approximately 2–3 h per UCB unit and isolated cells are highly enriched for an Oct‐4⁺ and SSEA‐4⁺ population of small Lin^?/CD45^?/CD133⁺ cells.