Expression Of T-cell Activation Markers Research Articles

P15.04 Genital tract cellular activation and inflammation associated with highly prevalent sexually transmitted infections and bacterial vaginosis in adolescent women at risk for hiv infection

Introduction The biological mechanisms underlying HIV risk in younger women is unclear. HIV is primarily transmitted across the genital mucosa and preferentially infects CD4 + T-cells. We investigated the influence of asymptomatic sexually transmitted infections (STIs) and bacterial vaginosis (BV) on CD4 + T-cell activation and inflammation in the genital tracts of adolescents from South Africa. Methods Cervical cytobrush mononuclear cells were isolated from 148 adolescents (16–22 years) from Cape Town, and expression of T-cell activation and proliferation markers (CD38, HLA-DR, Ki67, CCR5) was measured by FACs. Adolescents were screened for BV (Nugent) and STIs ( C. trachomatis , N. gonnorhoea, T. vaginalis , M. genitalium , HSV-2) by PCR. For comparison, 11 HIV-negative adult women were included. Concentrations of 48 cytokines, chemokines and growth factors were measured in matching menstrual cups by Luminex. Results Adolescents (median 18 years; IQR 17–20) had significantly higher frequencies of activated CD4 + T-cells (CD38 + , HLADR + , CD38 + HLADR + : each p C. trachomatis positive. Adolescents with an STI, despite these being asymptomatic, had higher frequencies of activated and proliferating T-cells compared to those with no STI/BV (CD4 + CD38 + HLADR + : p = 0.047; CD4 + Ki67 + : p = 0.020). Women positive for chlamydia had significantly higher frequencies of CD4 + CD38 + T-cells (p = 0.006). Women with both STIs and BV had the most pronounced increase in CD4 + T-cell activation (CD38 + : p = 0.002; CD38 + HLADR + : p = 0.001; Ki67+: p = 0.002). Higher cervical T-cell activation marker expression was directly associated with increased genital cytokine profiles. Conclusion Heightened levels of genital immune activation and inflammation found in South African adolescent females, partly due to the presence of asymptomatic STIs and BV could increase their risk for HIV infection.

PL05.2 Why sti-associated genital tract inflammation still matters in hiv transmission

Women in Africa, especially young women, have very high HIV incidence rates that cannot be fully explained by behavioural risks. In the setting of syndromic management for sexually transmitted infections (STIs) and bacterial vaginosis (BV), the influence of these, particularly asymptomatic infections, on CD4+ T cell activation and inflammation in the genital tracts of adolescents from South Africa urgently needs to be addressed. The influence of genital inflammation on HIV acquisition in this group will be discussed. Our study found that HIV seroconversion was associated with raised genital inflammatory cytokines (including chemokines MIP-1α, MIP-1β and IP-10). The risk of HIV acquisition was significantly higher in women with evidence of genital inflammation, defined by at least 5 of 9 inflammatory cytokines being raised [OR 3.2; 95% confidence interval 1.3–7.9]. Genital cytokine concentrations were persistently raised (for about one year before infection), with no readily identifiable cause despite extensive investigation of several potential factors, including STIs and systemic cytokines. Adolescents (median 18 years) had significantly higher frequencies of activated CD4+ T-cells (CD38+, HLADR+, CD38+HLADR+) from cervical cytobrushes than adults, although CCR5 expression was higher in adults. STIs and BV prevalence was very high in certain areas of South Africa, with 71% of adolescents having >1 STI and/or BV, and 42% being C. trachomatis positive. Adolescents with an STI had higher frequencies of activated and proliferating T-cells compared to those with no STI/BV. Higher cervical T-cell activation marker expression was directly associated with increased genital cytokine profiles. Our data suggests that elevated genital concentrations of HIV target cell-recruiting chemokines and a genital inflammatory profile contributes to the high risk of HIV acquisition in these African women. In adolescents, heightened levels of genital immune activation and inflammation, partly due to the presence of asymptomatic STIs/BV, could increase their risk for HIV infection.

도라지 추출액과 한약재 함유 도라지 추출액에 의한 면역세포 활성 표지유전자 발현 분석

Extracts of Platycodon grandiflorum have been reported to show anti-inflammatory, antioxidant, anti-metastatic, and hepato-protective effects. This study was designed to evaluate T-cell activation and M1/M2 differential macrophage activation by extracts of P. grandiflorum or P. grandiflorum containing various medicinal herbs. Using real-time RT-PCR, we analyzed expression levels of c-fos, and CD40L(T-cell activation markers) in splenocytes and iNOS, Ym1, and ARG1 in RAW 246.7 cells after treatment of CC (hot water extract of P. grandiflorum), MAEK (hot water extract of P. grandiflorum [82%]and six different plants), and HWAL (hot water extract of P. grandiflorum [7%] and eight different plants. The results showed that MAEK significantly elevated the expression of T-cell activation markers of splenocytes, with the c-fos gene activated more than 10-fold and the CD40L gene activated more than 6-fold. Although CD40L was significantly increased by CC and HWAL, the increase was only about 2-fold. In addition, CC and HWAL did not significantly activate the expression of the c-fos gene. On the other hand, CC elevated the M1 activation marker iNOS, and HWAL elevated the M2 activation marker Ym1 and ARG1 gene expression. In conclusion, MAEK could be used as an immune stimulant because of its ability to activate T cells (elicited c-fos and CD40L gene expression), whereas HWAL could serve as an anti-inflammatory agent because of its differential activation of M2 macrophages.

Immunoregulatory T cells, LFA-3 and HLA-DR in autoimmune thyroid diseases.

Aim:The aim of the present study was to examine the changes in the expression of T-cell activation markers, namely CD4+ CD25+ and CD8+ in patients with AITD, namely Graves’ disease and Hashimoto's thyroiditis as well as colloid nodular goitre. HLA-DR, LFA-3, and peripheral total lymphocytic count are also measured.Materials and Methods:We compared the expression of CD4, CD25, and CD8 surface markers in peripheral blood lymphocyte in Graves’ disease and Hashimoto's thyroiditis as autoimmune thyroid diseases, as well as colloid goitre in comparison with healthy controls. Also, LFA-3 and HLA-DR were measured in the same groups using three-color flow cytometry. Total lymphocytic count in peripheral blood, thyroid function tests, antithyroid antibodies were also included in the laboratory investigations. The total number of participants was 65. All were recruited from endocrine clinics in a tertiary care hospital in the southern region of Saudi Arabia. All participants underwent history taking, clinical examination, laboratory workup, and radiological investigations. Neck ultrasound, technecium pertechnetateψψ thyroid uptake, and fine-needle aspiration and cytology (FNAC) of the thyroid were done when indicated. The study was approved by the Hospital Research Isthics Committee and informed consents were obtained from all participants before enrollment in the study.Results:In comparison with thecontrol group, activation markers CD4, CD25, and CD8 were lower in the autoimmune thyroid diseases. Lymphocyte function antigen-3 (CD58) and total lymphocytic count were higher in the AIT diseases whereas HLA-DR was lower than that in the control group. The CD4/CD8 ratio was lower in the AITD compared with the healthy euthyroid subjects. No difference was found between patients with colloid nodular goitre and the healthy control in any of the study variables except for LFA-3 which was significantly higher in the colloid goitre group.Conclusion:Our findings indicate downregulation of CD4+ CD25+ Treg as well as CD8+ T cells in autoimmune thyroid diseases. Downregulation of suppressor T lymphocytes helps initiation, progression, and maintenance of the autoimmune thyroid diseases. Lower HLA-DR and higher CD58 in AITDs indicate their role in the expression of the autoantigen and its escape from the immune surveillance. High levels of LFA-3 in colloid goitre indicate that the autoimmune process needs interacting factors, and not only the high level of LFA-3.

Safety and immunogenicity of an adjuvanted protein therapeutic HIV-1 vaccine in subjects with HIV-1 infection: A randomised placebo-controlled study

The human immunodeficiency virus type-1 (HIV-1) vaccine candidate F4/AS01 has previously been shown to induce potent and persistent polyfunctional CD4+ T-cell responses in HIV-1-seronegative volunteers. This placebo-controlled study evaluated two doses of F4/AS01 1-month apart in antiretroviral treatment (ART)-experienced and ART-naïve HIV-1-infected subjects (1:1 randomisation in each cohort). Safety, HIV-1-specific CD4+ and CD8+ T-cell responses, absolute CD4+ T-cell counts and HIV-1 viral load were monitored for 12 months post-vaccination. Reactogenicity was clinically acceptable and no vaccine-related serious adverse events were reported. The frequency of HIV-1-specific CD4+ T-cells 2 weeks post-dose 2 was significantly higher in the vaccine group than in the placebo group in both cohorts (p<0.05). Vaccine-induced HIV-1-specific CD4+ T-cells exhibited a polyfunctional phenotype, expressing at least CD40L and IL-2. No increase in HIV-1-specific CD8+ T-cells or change in CD8+ T-cell activation marker expression profile was detected. Absolute CD4+ T-cell counts were variable over time in both cohorts. Viral load remained suppressed in ART-experienced subjects. In ART-naïve subjects, a transient reduction in viral load from baseline was observed 2 weeks after the second F4/AS01 dose, which was concurrent with a higher frequency of HIV-1-specific CD4+ T-cells expressing at least IL-2 in this cohort. In conclusion, F4/AS01 showed a clinically acceptable reactogenicity and safety profile, and induced polyfunctional HIV-1-specific CD4+ T-cell responses in ART-experienced and ART-naïve subjects. These findings support further clinical investigation of F4/AS01 as a potential HIV-1 vaccine for therapeutic use in individuals with HIV-1 infection.

OP0195 Genetic and Pharmacologic Inhibition of MST1 Blocks Lymphocyte Function and Protects Against Inflammation and Autoimmunity

BackgroundThe mammalian sterile 20-like kinase 1 (MST1) is a MAP4 kinase which acts upstream of a wide range of signal transduction pathways. It can modify signaling events mediated by Jnk,...

HIV and Tuberculosis co-infection impacts T-cell activation markers but not the numbers subset of regulatory T-cells in HIV-1 infected patients.

BackgroundTuberculosis (TB) has been shown to accelerate the clinical course of HIV infection, but the mechanisms by which this occurs are not well understood. Regulatory T-cells (Tregs) are known to dampen hyperactivation of the immune cells, but it remains unclear whether hyperactivation of T-cells in HIV infection is associated with a decrease of Tregs and what the effect Mycobacterium tuberculosis (MTB) co-infection has on T-cell activation and Tregs.ObjectivesIn this study, we aim to evaluate whether active TB is associated with the increased expression of T-cell activation markers and reduced number of Treg cells in HIV-1-infected patients.MethodsThis study was conducted on 69 subjects consisting of 20 HIV-infected patients, 20 HIV and MTB co-infected patients, 19 MTB-infected patients and 10 uninfected control subjects negative for both MTB and HIV. The frequencies of T-cell activation markers (CD38 and HLA-DR) and Treg cells (CD4+CD25+CD127-) were measured by flow cytometry.ResultsSignificantly higher expression of CD38 and HLA-DR on CD4+ and CD8+ T-cells was found in MTB and HIV co-infected patients compared with HIV-infected patients. However, no significant difference in the percentage of Treg cells was reported between HIV patients with TB and those without. The study also showed a negative correlation between regulatory T-cells frequency and CD4+ T-cell counts.ConclusionThese results suggest that TB enhances the expression of peripheral T-cell activation markers during HIV infection, whilst having no impact on the percentages of Treg cells.

CTLA4 Blockade Induces Frequent Tumor Infiltration by Activated Lymphocytes Regardless of Clinical Responses in Humans

CTLA4 blocking monoclonal antibodies provide durable clinical benefit in a subset of patients with advanced melanoma mediated by intratumoral lymphocytic infiltrates. A key question is defining whether the intratumoral infiltration (ITI) is a differentiating factor between patients with and without tumor responses. Paired baseline and postdosing tumor biopsy specimens were prospectively collected from 19 patients with metastatic melanoma, including 3 patients with an objective tumor response, receiving the anti-CTLA4 antibody tremelimumab within a clinical trial with primary endpoint of quantitating CD8(+) cytotoxic T-lymphocyte (CTL) infiltration in tumors. Samples were analyzed for cell density by automated imaging capture and further characterized for functional lymphocyte properties by assessing the cell activation markers HLA-DR and CD45RO, the cell proliferation marker Ki67, and the regulatory T-cell marker FOXP3. There was a highly significant increase in ITI by CD8(+) cells in biopsy samples taken after tremelimumab treatment. This included increases between 1-fold and 100-fold changes in 14 of 18 evaluable cases regardless of clinical tumor response or progression. There was no difference between the absolute number, location, or cell density of infiltrating cells between clinical responders and patients with nonresponding lesions that showed acquired intratumoral infiltrates. There were similar levels of expression of T-cell activation markers (CD45RO, HLA-DR) in both groups and no difference in markers for cell replication (Ki67) or the suppressor cell marker FOXP3. CTLA4 blockade induces frequent increases in ITI by T cells despite which only a minority of patients have objective tumor responses.

Impairment in Immunomodulatory Function of Mesenchymal Stem Cells from Multiple Myeloma Patients

Abnormality of immune regulation exists in multiple myeloma (MM). Mesenchymal stem cells (MSCs), a key regulator for immunomodulatory function, have decreased osteogenic potential in MM patients. Here we investigated the immunomodulatory function of MSCs from MM patients (MM-MSCs) and its relationship with decreased osteogenic potential. Real-time PCR was performed to detect the cytokines expressed in MM-MSCs (n = 22) and MSCs from normal donors (ND-MSCs, n = 11). Lymphocyte proliferative assay was used to detect the effect of MSCs on T cell proliferation. The effect of MSCs on T-cell cycle and T-cell activation markers expression were analyzed by flow cytometry. Flow cytometry and Western blot were used to detect apoptosis of T cells. Influence of T cells on osteogenic potential of MSCs was detected. MM-MSCs exhibited increased expression of TGF-β1, IL-6, IL-3, TNF-α and RANKL and decreased expression of TGF-β2, TGF-β3 and FasL. The inhibitory effect of MM-MSCs on T.cell proliferative ability was attenuated. ND-MSCs silence more T cells in G0/G1 phase than MM-MSCs. The apoptosis-promoting effect of MM-MSCs on T cells seemed to be dampened. Expression of T-cell activation markers was significantly inhibited by ND-MSCs. T cells from normal donors possessed the ability to promote osteoblastic differentiation of ND-MSCs, but this ability of T cells both directly from MM patients and co-cultured with MM-MSCs was impaired. MSCs from MM patients showed impaired immunoinhibitory capability on T cells, which in turn lose the ability to stimulate osteogenesis of MSCs.

In Vitro Differentiation of Adult Bone Marrow Progenitors into Antigen-Specific CD4 Helper T Cells Using Engineered Stromal Cells Expressing a Notch Ligand and a Major Histocompatibility Complex Class II Protein

A murine stromal cell line (OP9-DL1) expressing a notch ligand, Delta-like-1, has been shown to be able to drive the differentiation of both murine and human hematopoietic progenitors into T cells in vitro. Further studies showed that hematopoietic progenitors transduced by a retroviral vector to express a human CD8 T-cell receptor (TCR) followed by an OP9-DL1 monolayer coculture could generate antigen-specific cytotoxic T lymphocytes in vitro. It remains unknown if a similar method could be applied to produce CD4 helper T cells. In this report, we show that murine adult bone marrow (BM) cells transduced with an OT2 CD4 TCR and cocultured with OP9 stromal cells expressing Delta-like-1 can differentiate into antigen-specific CD4 T cells in vitro. These cells are capable of inducing the expression of T-cell activation markers and producing cytokines upon stimulation. We have also constructed a new stromal cell line (OP9-DL1-IA(b)) ectopically expressing a murine major histocompatibility complex class II protein, I-A(b), in OP9-DL1 cells. This new line could accelerate the development of TCR-transduced BM cells into CD4 T cells, resulting in cells with an improved capacity to respond to T-cell stimulation to secrete cytokines. Taken together, we demonstrate a general and potentially useful method to generate autologous antigen-specific CD4 helper T cells in vitro from easily accessible BM cells.

Nilotinib inhibits the Src‐family kinase LCK and T‐cell function in vitro

Dear Editor: In a recent issue of the Journal of Cellular and Molecular Medicine, it was reported that the tyrosine kinase inhibitor nilotinib (AMN-107, Tasigna®; Norvartis Pharmaceuticals, Basel, Switzerland), used for the treatment of chronic myeloid leukaemia, is able to inhibit the function of normal human T lymphocytes in vitro[1]. In addition, this group demonstrated nilotinib inhibits T-cell receptor (TCR) activation and the phosphorylation of signalling proteins involved in TCR activation. We have also investigated the effects of nilotinib on T cells and have expanded on the findings of Chen et al. by demonstrating that like imatinib [2], nilotinib is able to inhibit LCK, a Src-family kinase that plays a critical role in TCR activation [3]. Experiments were performed using normal human peripheral blood mononuclear cells (PBMCs) isolated by density centrifugation. Experimental use of human material was approved by the Royal Adelaide Hospital Ethics Committee and blood was collected with informed consent. 5’6 carboxyfluorescein diacetate succin-imidyl ester (CFSE) staining of human PBMCs was performed as described previously [4]. In the presence of varying concentrations of nilotinib and imatinib (Novartis Pharmaceuticals), T cells were stimulated with 10 μg/ml phytohaemagglutinin (PHA) or Concanavalin A (ConA) (Sigma, St. Louis, MO, USA) or directly using 75 ng/ml of an anti-CD3 antibody (Mabtech, Stockholm, Sweden). Following a 5-day incubation, the proliferation of T cells determined by analysing CFSE dye dilution in cells stained positive by an anti-CD3 PE antibody (BD Biosciences, San Jose, CA, USA) (Fig. 1A). T-cell data were then analysed by Modfit analysis program (Verity Software, Topsham, ME, USA) and the proliferation index determined for each sample. Graphing these values allowed IC50 values for each drug to be determined. Inhibition of LCK by nilotinib and imatinib was analysed by evaluating the effects of each drug on LCK phosphorylation of a substrate peptide. Active LCK kinase (Cell Signaling, Danvers, MA, USA) was incubated with varying concentrations of both drugs in a standard kinase reaction buffer containing a mix of unlabelled and γ32p labelled ATP and a Src-family kinase peptide substrate (Upstate Biotechnology, Lake Placid, NY, USA). The level of γ32pATP labelled substrate, and hence LCK activity, was determined by blotting reactions on p81 filter paper (Whatman, Kent, UK) and determining radioactivity of each sample using a bench top scintillation β-counter. Nilotinib inhibits T-cell proliferation and LCK activity. Following PHA stimulation, nilotinib was able to strongly inhibit the proliferation of T cells as determined by CFSE tracking (A). Using this CFSE data, the proliferation index at various drug concentrations was determined and used to calculate IC50 values for the inhibition of proliferation (B). Values represent the mean from five donors each analysed in different experiments and significant donor variability was seen as represented by large standard deviations. The effects of varying concentrations of nilotinib and imatinib on LCK kinase activity was determined and normalized to a percentage of maximum kinase activity when no drug was present allowing IC50 values to be determined (C). Data presented represent the mean of three independent experiments. Nilotinib inhibited T-cell proliferation at IC50's of 2–5 μM depending on the stimulus used (Fig. 1B). The IC50 values were roughly half that we observed with imatinib. We also observed a similar effect of nilotinib on T-cell activation marker expression and cytokine production (data not shown). LCK kinase activity was inhibited by nilotinib with an IC50 of 550 nM (Fig. 1C). Imatinib inhibited LCK at a similar concentration as reported in the literature [2] with an IC50 of 1250 nM, approximately twice the IC50 of nilotinib. In contrast to our findings, another group [5] found nilotinib to inhibit LCK weakly with an IC50 of 5200 nM. While this is somewhat less potent inhibition to that we obtained, the different IC50s could be due to technical differences in the kinase assays used. Abl has also been implicated in T-cell function [6–8] and it is possible that Abl inhibition by nilotinib may cause, or add to LCK blockade for the inhibitory effect on T-cell function. Nilotinib has 20-fold increased potency against Abl compared with imatinib [9] and we found it to have twice the potency against LCK. As nilotinib inhibited T cells approximately twice as strongly as imatinib, not 20 times as strongly, our data would suggest LCK inhibition may be the main mechanism by which the drug inhibits T-cell activation. Our findings agree well with those of Chen et al.[1] and expand on their results by demonstrating nilotinib inhibits the activity of the Src-family kinase LCK, and propose inhibition of LCK as the likely mechanism by which nilotinib interrupts TCR signalling and the function of T cells. We would like to thank Novartis for supplying the nilotinib used in these experiments. One of the authors, Professor Tim Hughes, is on the Advisory Board of Novartis Pharmaceuticals and also receives research funding from the company.

In vitro mitogen-stimulated T-cell from hepatitis C virus-positive liver transplantation candidates, increases T-cell activation markers and T-cell proliferation

The incidence of acute rejection is significantly higher in hepatitis C virus (HCV) liver-transplant patients than in patients who have received a graft for other liver diseases, i.e., mainly alcoholic cirrhosis. The aim of this study was to assess T-cell function, i.e., intralymphocyte cytokine expression (IL-2 and TNF-α), T-cell activation [i.e., transferrin receptor (CD71) and interleukin (IL)-2 α-chain (CD25) expression], and T-cell proliferation using a flow-cytometry whole-blood assay in patients waiting for a liver transplantation ( n = 49). Our data suggest that, in mitogen-stimulated T-cells, (i) intra-lymphocyte cytokine expression is significantly higher in patients with liver disease than in healthy volunteers ( n = 25); (ii) the expression of T-cell activation markers is decreased in patients with liver cirrhosis compared to healthy volunteers, and (iii) the expression of T-cell activation markers and T-cell proliferation are increased in patients with HCV infection ( n = 15) compared to those without HCV infection ( n = 34), particularly compared to patients with alcoholic liver disease ( n = 19). Circulating CD19-positive cells count was also significantly higher in HCV-positive patients. In conclusion, in vitro, mitogen-stimulated T-cell seem to induce a higher immune response in the blood from patients waiting for a liver transplant for HCV-related liver disease than those without HCV infection, and particularly those with alcoholic liver disease.

TISSUE‐SPECIFIC EXPRESSION OF B‐CELL TRANSLOCATION GENE 2 (BTG2) AND ITS FUNCTION IN T‐CELL IMMUNE RESPONSES IN A TRANSGENIC MOUSE MODEL

B-cell translocation gene 2 (BTG2) belongs to the anti-proliferative gene family. According to previous in vitro studies, BTG2 overexpression leads to delayed cell cycling. We investigated BTG2 expression during mouse ontogeny and its immune and circadian functions in this study. In situ hybridization showed that BTG2 was expressed at high levels in the central nervous system, liver, stomach, thymus, spleen, skin, adrenal gland, pituitary gland and salivary glands during embryonic days (E10-E17), postnatal days (P1 and P10) and adult stages. Expression was observed in organs and tissues from adult mice with and without a robust proliferation program. Thus, the gene might have important functions that are both related and unrelated to proliferation. BTG2 expression was induced after in vitro T-cell receptor stimulation in T cells using anti-CD3 antibodies. However, transgenic (Tg) mice with actin promoter-driven expression of BTG2 showed normal in vitro and in vivo T-cell responses, such as thymus development, T-cell activation marker expression, T-cell proliferation and migration, as well as in vivo delayed-type hypersensitivity reactions. Although BTG2 was expressed in the suprachiasmatic nucleus and pineal gland in the brain, BTG2 Tg mice had no abnormal circadian behavior. Our data on BTG2 expression during ontogeny provide useful clues for the further investigation of BTG2 function. Additional studies are warranted to examine its role in immune and other systems.

Tissue-specific expression of B-cell translocation gene 2 (BTG2) and its function in T-cell immune responses in a transgenic mouse model

B-cell translocation gene 2 (BTG2) belongs to the anti-proliferative gene family. According to previous in vitro studies, BTG2 overexpression leads to delayed cell cycling. We investigated BTG2 expression during mouse ontogeny and its immune and circadian functions in this study. In situ hybridization showed that BTG2 was expressed at high levels in the central nervous system, liver, stomach, thymus, spleen, skin, adrenal gland, pituitary gland and salivary glands during embryonic days (E10-E17), postnatal days (P1 and P10) and adult stages. Expression was observed in organs and tissues from adult mice with and without a robust proliferation program. Thus, the gene might have important functions that are both related and unrelated to proliferation. BTG2 expression was induced after in vitro T-cell receptor stimulation in T cells using anti-CD3 antibodies. However, transgenic (Tg) mice with actin promoter-driven expression of BTG2 showed normal in vitro and in vivo T-cell responses, such as thymus development, T-cell activation marker expression, T-cell proliferation and migration, as well as in vivo delayed-type hypersensitivity reactions. Although BTG2 was expressed in the suprachiasmatic nucleus and pineal gland in the brain, BTG2 Tg mice had no abnormal circadian behavior. Our data on BTG2 expression during ontogeny provide useful clues for the further investigation of BTG2 function. Additional studies are warranted to examine its role in immune and other systems.

Marginal Zone B-Cell Depletion Impairs Murine Host Defense againstBorrelia burgdorferiInfection

Marginal zone B (MZB) cells are a B-cell subset that produces T-cell-independent antibodies to blood-borne antigens. In this study, we examined the effects of MZB cell depletion on the immune response to the Lyme disease spirochete Borrelia burgdorferi, an extracellular pathogen for which T-cell-independent antibody is an important host defense. MZB cell depletion of C3H/HeJ mice using monoclonal antibody to LFA-1 and alpha(4)beta(1) integrins reduced B. burgdorferi-specific immunoglobulin M (IgM) titers, enhanced pathogen burden, and led to more severe arthritis assessed within the first 2 weeks of infection. In addition, MZB cell-depleted mice had reduced levels of B. burgdorferi-specific IgG, which correlated with diminished splenic CD4+ T-cell-activation, proliferation, and cytokine production. Passive transfer of immune mouse serum from infected control mice into infected MZB cell-depleted mice reduced pathogen burden but did not alter the expression of T-cell activation markers on splenic CD4+ T cells. These findings demonstrate that MZB cells not only are a source of pathogen-specific IgM important for limiting spirochete burden and pathology but also play a prominent role in the priming of splenic T-cell responses to a blood-borne pathogen.

Activation and Maturation of Alloreactive CD4-Independent, CD8+ Cytolytic T Cells

The goal of this study was to determine the in vivo conditions that promote activation of the (CD4-independent) CD8+ T cell-mediated rejection pathway. We have previously noted that hepatocellular but not islet allografts readily activate this rejection pathway. In the current study, we utilized these two cell transplant models to investigate whether differences in host cell recruitment to the graft site, expression of T-cell activation markers by CD8+ graft infiltrating cells (GICs), and/or development of delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte cell-mediated effector functions could account for the differential transplant outcomes. The collective results demonstrate that recruitment of CD8+ T cells to the site of transplant, CD103 or CD69 expression on CD8+ GICs, and activation of alloreactive DTH responses are insufficient to initiate CD4-independent, CD8-dependent transplant rejection. Instead, rejection by alloreactive (CD4-independent) CD8+ T cells correlated with expression of CD25, CD154 and CD43 by CD8+ GICs, in vitro alloproliferation by recipient CD8+ T cells, and the development of in vivo allospecific cytolytic effector function. These results suggest that tissue-derived factors influence the activation and maturation of (CD4-independent) CD8+ T cells into cytolytic effectors, which correlates with transplant rejection.

The role of leukemia-derived B7-H1 (PD-L1) in tumor–T-cell interactions in humans

Expression of the B7 homolog B7-H1 (PD1-Ligand) has been proposed to enable tumor cells to evade immune surveillance. Recently, B7-H1 on murine leukemia cells was reported to mediate resistance to cytolytic T-cell destruction. We here investigate the expression and function of the B7 homolog B7-H1 in human leukemia. Leukemia cells from 30 patients and 9 human leukemia cell lines were investigated for B7-H1 expression by flow cytometry. Functional relevance of B7-H1 for tumor-immune interactions was assessed by coculture experiments using purified, alloreactive CD4 and CD8 T cells in the presence of a neutralizing anti-B7-H1 antibody. Significant B7-H1 expression levels on leukemia cells were detected in 17 of 30 patients and in eight of nine cell lines. In contrast to various other tumor entities and the data reported from a murine leukemia system, no significant inhibitory effect of leukemia-derived B7-H1 on CD4 and CD8 cytokine production (IFN-gamma, IL-2), proliferation or expression of T-cell activation markers (ICOS, CD69) was observed. Furthermore, in the presence of neutralizing B7-H1 antibody (mAb 5H1) occurred no significant changes in T cell IFN-gamma or IL-2 production or proliferation. Our data demonstrate that leukemia-derived B7-H1 seems to have no direct influence on T-cell activation, proliferation, and cytokine production in humans. Further experiments are warranted to delineate factors and characterize yet-unidentified B7-H1 receptor(s) that determine inhibitory and stimulatory functions of B7-H1 in human leukemia.

Immunophenotypic patterns of T-cell activation in neuroinflammatory diseases

We aimed to gain insights into the pathogen-specific differences in early adaptive immune responses following central nervous system infections with Borrelia burgdorferi and viral pathogens by studying the immunophenotypic patterns of T-cell activation. Moreover, we wished to determine whether the expression of T-cell activation markers reflects disease activity in multiple sclerosis (MS). Proportions of cerebrospinal fluid T-cells expressing the markers HLA-DR, CD25 and CD38 were determined in patients with MS (n = 40), acute viral meningomyeloradiculoneuritis (VID, n = 26), early neuroborreliosis (NB, n = 23) and non-inflammatory neurologic diseases (n = 51) by using flow cytometry. In relapsing-remitting MS, disease activity was assessed by clinical examination and magnetic resonance imaging. For each of the surface markers that were examined, significant differences in T cell proportions were found between patient groups. The proportion of HLA-DR+ T cells was higher and that of CD25+ T cells lower in NB compared with VID. These differences were attributable only to the early phase of the disease (< or = 6 days after symptom onset). Among MS patients, there was a trend for higher proportions of T cells expressing activation markers in patients with gadolinium-enhancing lesions. The decreased CD25 expression in NB may reflect immunomodulatory effects of B. burgdorferi facilitating persistent infection. Larger prospective studies of T-cell activation markers for ascertaining the association between cellular markers and clinical surrogates of disease activity in MS are warranted.

T-Cell Activation Marker Expression on Tumor-infiltrating Lymphocytes as Prognostic Factor in Cutaneous Malignant Melanoma

T-Cell Activation Marker Expression on Tumor-infiltrating Lymphocytes as Prognostic Factor in Cutaneous Malignant Melanoma

The Role of Leukemia Derived B7-H1 in Tumor-T-Cell Interactions in Humans.

Objective: Expression of the B7-homologue B7-H1 (PD1-Ligand) has been proposed to enable tumor cells to evade immune surveillance. Recently, B7-H1 on murine leukemia cells was reported to mediate resistance to cytolytic T-cell destruction. In this study we investigated the expression and functional role of the B7-homologue B7-H1 in human leukemia.Patients and Methods: Leukemia cells from 20 patients and 9 human leukemia cell lines were investigated for B7-H1 expression by flow cytometry. Functional relevance of B7-H1 for tumor-immune interactions was assessed by coculture experiments using purified, alloreactive CD4 and CD8 T-cells in the presence of a neutralizing anti-B7-H1 antibody.Results: Significant B7-H1 expression levels on leukemia cells were detected in 13 of 20 patients and in 8 of 9 cell lines. In contrast to various other tumor entities and the data reported from a murine leukemia system we did not observe any significant inhibitory effect of leukemia-derived B7-H1 on CD4 and CD8 cytokine production (IFN-g, IL-2) or expression of T-cell activation markers (ICOS, CD69). In the presence of a neutralizing B7-H1 antibody (mAb 5H1) no significant changes in T cell IFN-g or IL-2 production were observed.Conclusions: Our data demonstrate that leukemia-derived B7-H1 seems to have no direct influence on T-cell activation and cytokine production in humans. Further experiments are warranted to delineate factors and characterize yet unidentified B7-H1 receptor(s) that determine inhibitory and stimulatory functions of B7-H1 in human leukemia.