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The antiergotypic response leads to the formation of effector T cells able to eliminate activated lymphocytes independently of their antigenic specificity, since the targets of these cells are molecules produced during cell activation (ergotopes). In this paper, we describe the level of expression of the ergotope-associated markers CD25, HSP60, and HLA-DR by the T lymphocytes isolated from the blood of atopic dermatitis patients immediately after isolation and after cultivation. After 10-day cultivation in the presence of anti-CD3 antibodies and IL-2, the expression levels of early and late activation markers in T cells have changed: the shares of CD25-positive CD4 and CD8 lymphocytes increase to 68 and 47%, respectively, and the share of HLA-DR-positive cells increases to 26 and 33%. The density of HLA-DR molecules on the surface of activated T cells increases more than fivefold. Almost all T cells before and after cultivation express 60 kDa heatshock protein (HSP60); however, the CD4 cells activated in vitro contain more HSP60 molecules than do the in vitro-activated CD8 cells and the CD4 cells of peripheral blood. Thus, the T cells of atopic-dermatitis patients have the status of activated cells because they express sufficient amounts of early and late activation markers; presumably, they can enhance the induction of antiergotypic response when administered to patients. Taking into account that antiergotypic regulation acts on activated T cells independently of their antigenic specificity, immunotherapy utilizing autologous activated T lymphocytes can be of interest as a method for targeted action on pathogenetic components of atopic dermatitis. [ABSTRACT FROM AUTHOR]

Freshly isolated B cells from chronic lymphocytic leukaemia patients (B-CLL) have been previously shown to induce a strong proliferative response and high levels of NK-like activity in lymphocytes from healthy donors. The present paper deals with the origin, mitotic state, target spectrum and cell surface phenotype of the NK-like effectors generated after stimulation with B-CLL. Experiments using large granular lymphocytes (LGL) and T cells as responders demonstrated that most of the precursors of the newly generated NK-like effectors express the CD3 antigen. The induction of NK-like activity paralleled cell activation, as judged by blast transformation, thymidine uptake and appearance of cell surface activation markers. The newly generated NK-like effectors displayed a T cell phenotype and a broader target repertoire than native NK cells. [ABSTRACT FROM AUTHOR]

Background: Current blood-based diagnostic tools for TB are insufficient to properly characterize the distinct stages of TB, from the latent infection (LTBI) to its active form (aTB); nor can they assess treatment efficacy. Several immune cell biomarkers have been proposed as potential candidates for the development of improved diagnostic tools. Objective: To compare the capacity of CD27, HLA-DR, CD38 and Ki-67 markers to characterize LTBI, active TB and patients who ended treatment and resolved TB. Methods: Blood was collected from 45 patients defined according to clinical and microbiological criteria as: LTBI, aTB with less than 1 month of treatment and aTB after completing treatment. Peripheral blood mononuclear cells were stimulated with ESAT-6/CFP-10 or PPD antigens and acquired for flow cytometry after labelling with conjugated antibodies against CD3, CD4, CD8, CD27, IFN-γ, TNF-α, CD38, HLA-DR, and Ki-67. Conventional and multiparametric analyses were done with FlowJo and OMIQ, respectively. Results: The expression of CD27, CD38, HLA-DR and Ki-67 markers was analyzed in CD4+ T-cells producing IFN-γ and/or TNF-α cytokines after ESAT-6/CFP-10 or PPD stimulation. Within antigen-responsive CD4+ T-cells, CD27− and CD38+ (ESAT-6/CFP-10-specific), and HLA-DR+ and Ki-67+ (PPD- and ESAT-6/CFP-10-specific) populations were significantly increased in aTB compared to LTBI. Ki-67 demonstrated the best discriminative performance as evaluated by ROC analyses (AUC > 0.9 after PPD stimulation). Data also points to a significant change in the expression of CD38 (ESAT-6/CFP-10-specific) and Ki-67 (PPD- and ESAT-6/CFP-10-specific) after ending the anti-TB treatment regimen. Furthermore, ratio based on the CD27 median fluorescence intensity in CD4+ T-cells over Mtb-specific CD4+ T-cells showed a positive association with aTB over LTBI (ESAT-6/CFP-10-specific). Additionally, multiparametric FlowSOM analyses revealed an increase in CD27 cell clusters and a decrease in HLA-DR cell clusters within Mtb-specific populations after the end of treatment. Conclusion: Our study independently confirms that CD27−, CD38+, HLA-DR+ and Ki-67+ populations on Mtb-specific CD4+ T-cells are increased during active TB disease. Multiparametric analyses unbiasedly identify clusters based on CD27 or HLA-DR whose abundance can be related to treatment efficacy. Further studies are necessary to pinpoint the convergence between conventional and multiparametric approaches. [ABSTRACT FROM AUTHOR]

Cancer immunotherapy is an evolving and promising cancer treatment that takes advantage of the body's immune system to yield effective tumor elimination. Importantly, immunotherapy has changed the treatment landscape for many cancers, resulting in remarkable tumor responses and improvements in patient survival. However, despite impressive tumor effects and extended patient survival, only a small proportion of patients respond, and others can develop immune-related adverse events associated with these therapies, which are associated with considerable costs. Therefore, strategies to increase the proportion of patients gaining a benefit from these treatments and/or increasing the durability of immune-mediated tumor response are still urgently needed. Currently, measurement of blood or tissue biomarkers has demonstrated sampling limitations, due to intrinsic tumor heterogeneity and the latter being invasive. In addition, the unique response patterns of these therapies are not adequately captured by conventional imaging modalities. Consequently, non-invasive, sensitive, and quantitative molecular imaging techniques, such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) using specific radiotracers, have been increasingly used for longitudinal whole-body monitoring of immune responses. Immunotherapies rely on the effector function of CD8+ T cells and natural killer cells (NK) at tumor lesions; therefore, the monitoring of these cytotoxic immune cells is of value for therapy response assessment. Different immune cell targets have been investigated as surrogate markers of response to immunotherapy, which motivated the development of multiple imaging agents. In this review, the targets and radiotracers being investigated for monitoring the functional status of immune effector cells are summarized, and their use for imaging of immune-related responses are reviewed along their limitations and pitfalls, of which multiple have already been translated to the clinic. Finally, emerging effector immune cell imaging strategies and future directions are provided. [ABSTRACT FROM AUTHOR]

Type 1 diabetes (T1D) is an autoimmune disease that is characterized by the specific destruction of insulin-producing pancreatic cells. Invariant natural killer b T (iNKT) cells have been associated with development of T1D. Class I MHC- restricted T cell-associated molecule (CRTAM) is expressed on activated iNKT, CD8+, and CD4+ T cells, and it is associated with the pro-inflammatory profiles of these cells. Crtam gene expression in CD3+ lymphocytes from non-obese diabetic (NOD) mice is associated with T1D onset. However, expression of CRTAM on T cells from patients with T1D has not yet been evaluated. We compared iNKT cell (CD3+Vα24+Vβ11+) numbers and CRTAM expression in a Mexican population with recent-onset T1D and their first-degree relatives with control families. Remarkably, we found lower iNKT cell numbers in T1D families, and we identified two iNKT cell populations in some of the families. One iNKT cell population expressed high iTCR levels (iNKThi), whereas another expressed low levels (iNKTlo) and also expressed CRTAM. These findings support a probable genetic determinant of iNKT cell numbers and a possible role for these cells in T1D development. This study also suggests that CRTAM identifies recently activated iNKT lymphocytes. [ABSTRACT FROM AUTHOR]

Many techniques are being used to examine the status of circulating human basophils including the enhanced expression of a variety of cell surface proteins. There is accumulating evidence that there are at least two compartments containing these activation marker proteins but there are only some indications for the signaling requirements for each of the compartments. This study began with published reports by other investigators who potentially dissociated CD63 expression from anaphylactic degranulation with the p38 inhibitor, SB203580, a possible falsification of a previously proposed hypothesis regarding CD63 expression. To explore the signaling requirements for CD63, a variety of pharmacological agents were used to inhibit or enhance 4 endpoints of basophil activation. First, it was found that inhibition of both histamine release and CD63 expression with SB203580 was concordant. But it was also found that this agent had no effect on increased expression of CD203c and CD11b. Actin polymerization inhibitors caused marked enhancement of CD63 expression (concordant with their effects on degranulation) with no effect on expression of CD203c and CD11b. The third generation syk inhibitor, NVP-QAB205, showed a 5-fold lower potency for inhibiting expression of CD203c and CD11b than for CD63. Finally, while desensitization of CD11b and CD203c expression occurs, it is slower than desensitization of the CD63 response. Taken together, these various observations demonstrate a marked difference in the early signaling requirements for the CD11b/CD203c compartment and CD63 degranulation and provide support for the hypothesis that CD11b and CD203c reside in a similar compartment. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Purpose: Particle radiations could significantly impact astronaut health during space missions. This study quantified the effects of iron ion radiation on lymphocytes in two strains of mice differing in susceptibility to radiation-induced acute myeloid leukemia (AML) and thymic lymphoma (TL): C57BL/6 (AML resistant, TL sensitive) and CBA/Ca (AML sensitive, TL resistant). Materials and methods: The animals ( n = 60/strain) were irradiated with 56Fe26+ (1 GeV) to total doses of 0, 0.5, 2 and 3 Gray (Gy) at an average dose rate of 1 Gy/min and euthanised on days 4 and 30 thereafter; blood, spleen, and bone marrow were collected for flow cytometry analyses. Cells expressing the following molecules were quantified: Cluster of differentiation (CD) 4, CD8, CD25, CD34, CD71, B220 (isoform of CD45 on B cells), NK1.1 (marker on natural killer or NK cells, C57B mice), panNK (marker on NK cells, CBA mice), and Sca1 (stem cell antigen 1). Results: Exposure to radiation resulted in different distribution patterns in lymphocyte populations and leukocytes expressing activation and progenitor markers in the two mouse strains. Significant main effects were dependent upon strain, as well as radiation dose, body compartment, and time of assessment. Especially striking differences were noted on day 4 after 3 Gy irradiation, including in the CD4:CD8 ratio [blood, C57 (2.83 ± 0.25) vs. CBA (6.19 ± 0.24); spleen, C57 (2.29 ± 0.12) vs. CBA (4.98 ± 0.22)], %CD25+ mononuclear cells in bone marrow [C57 (5.62 ± 1.19) vs. CBA (12.45 ± 0.93)] and %CD34+Sca1+ cells in bone marrow [CD45l° gate, C57 (2.72 ± 0.74) vs. CBA (21.44 ± 0.73)]. Conclusion: The results show that genetic background, as well as radiation dose and time post-exposure, had a profound impact on lymphocyte populations, as well as other leukocytes, after exposure to iron ion radiation. [ABSTRACT FROM AUTHOR]

The accelerated migration of Langerhans cells (LCs) out of the epidermis and up-regulation of maturation markers, upon treatment with subtoxic concentrations of chemicals, were used as the criteria to determine the potential of allergenic chemicals capable of inducing a hapten-specific delayed-type hypersensitivity reaction. Here we report the findings of a study in which seven chemicals, coded and tested in a blind fashion, were classified as contact allergens or non-allergens using the human organotypic skin explant culture (hOSEC) model. All chemicals that were identified as a contact sensitizer on decoding induced a definite decrease in the number of CD1a and HLA-DR-positive epidermal LCs in the epidermis of the skin explants, as determined by both semiquantitative immunohistochemistry and quantitative flow cytometric analysis. A significant increase in the number of CD83+ cells was accompanied by up-regulation of activation molecules in the epidermis of hOSEC exposed specifically to contact allergens. In contrast, there were only minor alterations in epidermal LC numbers, expression of CD83 and other activation markers by LCs when the biopsies were treated with non-toxic concentrations of non-allergenic irritants and vehicles. The data suggest that an increased epidermal LC migration and maturation accompanied by increased expression of activation markers could be used as end-point determinants to screen allergens in a non-animal alternative hOSEC model. [ABSTRACT FROM AUTHOR]

Abstract: Objective: To examine laboratory markers of platelet activation, thrombin generation and fibrinolysis in women with sickle cell disease (SCD) using the combined oral contraceptive pill (COCP), progestogen only (PO) contraception and non-hormonal contraception. Design: A prospective observational study set in two teaching hospitals in the London region. Method: Forty-four women with SCD in steady haematological state using differing hormonal contraception were recruited and venesection was performed at standardised times for the measurements of markers of platelet activation, thrombin generation and fibrinolysis. Outcome measures: Prothrombin fragment1+2 , plasmin α 2 antiplasmin complexes, platelet factor 4 (PF-4), β -thromboglobulin and free protein S antigen (PS-Ag). Results: PS-Ag was decreased and PF-4 increased in all women while the other haemostatic variables were within normal reference ranges. However, there was no statistically significant differences in the measurements of all the haemostatic variables between the three groups of sickle cell women (Kruskal–Wallis, P > 0.05). Conclusion: There is anxiety about prescribing the COCP in women with SCD based on the assumption that risk of venous thromboembolism may be compounded by the underlying disease process that occurs with these women. The observed data suggest that SCD women who use the COCP have haemostatic markers which are not statistically different compared with similar women who use PO contraception or non-hormonal contraception. However, a randomized interventional trial would be necessary to evaluate further the safety aspect of COCP use in this group of women. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) is characterized by B cell hyperactivity and the production of autoantibodies, some of which (antibodies to dsDNA) are thought to be pathogenic. T helper cells drive the production of autoantibodies and the aim of this study is to characterize phenotypically a subpopulation of T cells (the CD3[sup +]CD4[sup -]CD8[sup -], double negative (DN) T cells) previously identified as helping to enhance anti-DNA antibodies in patients with SLE. Data were obtained using FACS staining of DN T cells that had been purified from PBMCs by magnetic bead separation. The percentage of TCR αβ[sup +] DN T cells was found to be significantly higher in patients with SLE as compared with controls (P=0.02), although there was no significant increase in total percentage of DN T cells, which includes TCR γδ[sup +] cells. Activation markers HLA-DR and CD69, the costimulatory molecule CD28 and CTLA-4 were all expressed on the surface of a higher percentage of DN T cells in patients with SLE than in patients with rheumatoid arthritis (RA) or healthy controls (HC). More DN T cells from patients with SLE were of CD45RA phenotype than was found in controls, while CD45RO-expressing cells were reduced. In addition, DN T cells from patients with SLE expressed significantly higher levels of HLA-DR (P=0.006), CD28 (P=0.05), CTLA4 (P=0.03) and CD45RA (P=0.05) on the cell surface than those from the CD4/8 population. Correlation of expression of the markers measured with various parameters of disease activity and severity showed that high levels of HLA-DR expression correlated with high circulating serum C3 (>0.9IU/ml), indicating that an activated phenotype is consistent with severe disease. [ABSTRACT FROM AUTHOR]

Background Antigen-specific cell lines or clones are often used as models of drug-specific allergy. However, cloning procedures are time consuming, and the repeated antigen stimulation cycles as well as the addition of various growth enhancers may affect the in vivo relevance of these systems. Objective Using bacampicillin-allergic subjects, we wanted to investigate the applicability of primary recall in vitro lymphocyte responses to characterize type I and type IV allergy. The sensitivity and specificity of LTT (Lymphocyte transformation test), when used as an in vitro diagnostic tool, were also assessed. Methods A total of 39 patients with symptoms of type I (rhinitis) or type IV (allergic contact dermatitis, ACD) allergy following occupational exposure to bacampicillin, were included. Ten individuals without penicillin allergy or occupational exposure to bacampicillin served as controls. All subjects were LTT tested. Four patients with rhinitis and two patients with ACD were available for studying the immunophenotype and the TCR-Vβ repertoire of bacampicillin induced lymphoblasts as well as the cytokine profiles and expression of the activation markers CD23 and CD134 in primary PBMC cultures. Results LTT was positive in 87% and at least one of the skin tests was positive in 85% of the patients with allergic symptoms. 69% of the patients with type I allergies were patch test-positive. Results from LTT and skin test correlated in 87% of the cases. The combined sensitivity of LTT and skin tests was 92%. The specificity of LTT was 90% in healthy controls. Bacampicillin induced lymphoblasts were mainly CD4 + in both ACD and rhinitis patients. The TCR-Vβ profiles of the predominant CD4 + lymphoblasts were heterogeneous with individual skewing towards Vβ2, Vβ3, Vβ5.1 and/or Vβ14. An increased expression of IFNγ was detected in bacampicillin treated PBMC cultures from the ACD but not from rhinitis patients. IL-5 was detected in... [ABSTRACT FROM AUTHOR]

Bacterial genomic DNA, plasmid DNA (pDNA) and synthetic oligodeoxynucleotides (ODN) containing immunostimulatory DNA sequences (ISS) have been proposed to foster a Th1 response via the release of type 1 cytokines from macrophages, dendritic cells, NK cells and B cells. In this study, we show that ISS-enriched DNA up-regulates a distinct profile of cell surface molecules on macrophages and B cells in vitro and in vivo. ISS-ODN and ISS-containing pDNA enhanced the expression of antigen presentation molecules (MHC class I and II), co-stimulatory molecules (B7-1, B7-2 and CD40), cytokine receptors (IFN-γ receptor and IL-2 receptor), an adhesion molecule (ICAM-1) and an Fc receptor (Fcγ receptor) on murine B cells or bone marrow-derived macrophages. The increased expression of these surface molecules is seen in purified cell populations and is largely independent of the effects of type 1 cytokines. Splenic antigen-presenting cells stimulated with ISS-ODN in vivo efficiently activate naive T cells and bias their differentiation toward a Th1 phenotype in vitro . Thus, the induction of both type 1 cytokines and a distinct profile of cell surface molecules contributes to the potent immunostimulatory effects of ISS-containing DNA on innate and adaptive immunity. [ABSTRACT FROM PUBLISHER]

Several lines of evidence argue in favour of an involvement of T cells in the pathogenesis of Wegener's granulomatosis (WG). These include the presence of highly specific IgG autoantibodies to proteinase 3, perivascular T-cell infiltrates and elevated amounts of soluble interleukin-2 (IL-2) receptors in patient's serum. In order to further address this question we evaluated by double immunoflourescence and flow cytometry the expression of several cell surface molecules associated with T-cell activation. As compared to healthy controls ( n=15), the CD4 subset was significantly diminished, while the percentage of CD8 T cells was elevated in WG patients ( n=24). Within the CD4 T-cell subset we found a highly significant increase in activation/memory markers (CD25, CD29, HLA-DR). Within the CD8 T-cell subset the expression of CD11b, CD29 and CD57 was significantly elevated, while the expression of VD28 was reduced. The use of 10 Vβ-, 1 Vα-and 1 Vγ-specific monoclonal reagents failed to reveal any significant bias in the peripheral T-cell receptor V-gene repertoire of WG patients. There was also no correlation between T-cell activation markers and laboratory parameters [C-reactive protein (CRP), ESR], disease duration or therapy. A significant correlation was found only for the degree of organ involvement and the increase in CD4 T cells coexpressing HLA-DR, as well as the increase in CD57 expression on CD8 T cells. In conclusion, both CD4 and CD8 T-cell subsets were activated in WG. Cytotoxic CD8 CD57 CD11b CD28 T cells may directly contribute to damage of vascular endothelium. [ABSTRACT FROM AUTHOR]