T-cell Proliferative Capacity Research Articles

CD160-associated CD8 T-cell functional impairment is independent of PD-1 expression.

Expression of co-inhibitory molecules is generally associated with T-cell dysfunction in chronic viral infections such as HIV or HCV. However, their relative contribution in the T-cell impairment remains unclear. In the present study, we have evaluated the impact of the expression of co-inhibitory molecules such as 2B4, PD-1 and CD160 on the functions of CD8 T-cells specific to influenza, EBV and CMV. We show that CD8 T-cell populations expressing CD160, but not PD-1, had reduced proliferation capacity and perforin expression, thus indicating that the functional impairment in CD160+ CD8 T cells may be independent of PD-1 expression. The blockade of CD160/CD160-ligand interaction restored CD8 T-cell proliferation capacity, and the extent of restoration directly correlated with the ex vivo proportion of CD160+ CD8 T cells suggesting that CD160 negatively regulates TCR-mediated signaling. Furthermore, CD160 expression was not up-regulated upon T-cell activation or proliferation as compared to PD-1. Taken together, these results provide evidence that CD160-associated CD8 T-cell functional impairment is independent of PD-1 expression.

Blockade of the programmed death-1 pathway restores sarcoidosis CD4(+) T-cell proliferative capacity.

Effective therapeutic interventions for chronic, idiopathic lung diseases remain elusive. Normalized T-cell function is an important contributor to spontaneous resolution of pulmonary sarcoidosis. Up-regulation of inhibitor receptors, such as programmed death-1 (PD-1) and its ligand, PD-L1, are important inhibitors of T-cell function. To determine the effects of PD-1 pathway blockade on sarcoidosis CD4(+) T-cell proliferative capacity. Gene expression profiles of sarcoidosis and healthy control peripheral blood mononuclear cells were analyzed at baseline and follow-up. Flow cytometry was used to measure ex vivo expression of PD-1 and PD-L1 on systemic and bronchoalveolar lavage-derived cells of subjects with sarcoidosis and control subjects, as well as the effects of PD-1 pathway blockade on cellular proliferation after T-cell receptor stimulation. Immunohistochemistry analysis for PD-1/PD-L1 expression was conducted on sarcoidosis, malignant, and healthy control lung specimens. Microarray analysis demonstrates longitudinal increase in PDCD1 gene expression in sarcoidosis peripheral blood mononuclear cells. Immunohistochemistry analysis revealed increased PD-L1 expression within sarcoidosis granulomas and lung malignancy, but this was absent in healthy lungs. Increased numbers of sarcoidosis PD-1(+) CD4(+) T cells are present systemically, compared with healthy control subjects (P < 0.0001). Lymphocytes with reduced proliferative capacity exhibited increased proliferation with PD-1 pathway blockade. Longitudinal analysis of subjects with sarcoidosis revealed reduced PD-1(+) CD4(+) T cells with spontaneous clinical resolution but not with disease progression. Analogous to the effects in other chronic lung diseases, these findings demonstrate that the PD-1 pathway is an important contributor to sarcoidosis CD4(+) T-cell proliferative capacity and clinical outcome. Blockade of the PD-1 pathway may be a viable therapeutic target to optimize clinical outcomes.

Candida skin test reagent as a novel adjuvant for a human papillomavirus peptide-based therapeutic vaccine

A vaccine adjuvant that can effectively promote cell-mediated immunity is currently not available. Because of the ability of a Candida skin test reagent injection to induce common wart regression, our group is using it as a novel adjuvant in a clinical trial of a peptide-based human papillomavirus therapeutic vaccine. The goal of this current study was to investigate the mechanisms of how Candida enhances the vaccine immune responses. Maturation effects on Langerhans cells, capacity to proliferate T-cells, expression of cytokines and pattern recognition receptors by Langerhans cells, and ability to induce Th1, Th2, and Th17 responses were investigated in healthy subjects. The vaccine, human papillomavirus peptides with Candida, demonstrated partial maturation effects on Langerhans cells indicated by significantly up-regulated CD40 (p=0.00007) and CD80 (p<0.00001) levels, and showed T-cell proliferative capacity (p<0.00001) when presented by Langerhans cells in vitro. Interestingly, the maturation effects were due to the peptides while Candida was responsible for the T-cell proliferation. The cytokine profile (IL-1β, IL-6, IL-8, IL-10, IL-12p40, IL-23Ap19, IFN-γ and TNF-α) of Langerhans cells treated with the vaccine or Candida alone showed that IL-12p40 mRNA was most frequently induced, and IL-12p70 protein was detected in the supernatants. The presence of pattern recognition receptors known to associate with Candida albicans (DC-SIGN, dectin-1, dectin-2, galectin-3, mincle, mannose receptor, Toll-like receptors-1, 2, 4, 6 and 9) were demonstrated in all subjects. On the other hand, the induction of Th1 response demonstrated by IFN-γ secretion by CD4 cells stimulated with the vaccine or Candida pulsed Langerhans cells was demonstrated only in one subject. In summary, the Langerhans cell maturation effects of the vaccine were due to the peptides while the T-cell proliferative capacity was derived from Candida, and the most frequently induced cytokine was IL-12.

Abstract IA6: Immune escape mechanisms in lymphoid malignancies.

Abstract Certain lymphoid malignancies utilize potent mechanisms to evade host anti-tumor immune responses including the AP-1-dependent overexpression of Galectin-1 (Gal1) and the genetic amplification and overexpression of the PD-1 ligands. Gal1 recognizes specific cell surface glycans (Gal1 β1 GlcNAc [N-acetyllactosamine] units on the branches of N- or O-linked glycans) on receptors such as CD45, CD43 and CD7 and induces the apoptosis of select T cell subsets – Th1, Th17 and cytotoxic T cells. Th2 cells and regulatory T (Treg) cells have different patterns of sialylation of cell surface glycoproteins, lack Gal1 ligands and resist Gal1-induced death. We recently found that malignant Reed-Sternberg (RS) cells in classical Hodgkin Lymphoma (cHL) and EBV-transformed malignant B cells in post-transplant lymphoproliferative disorders (PTLD) overexpress the secreted carbohydrate-binding lectin, Galectin-1 (Gal1). Gal1 overexpression by Hodgkin RS cells and EBV-transformed B cells promotes the immunosuppressive Th2/Treg-predominant, Th1/cytotoxic T cell-deficient micro-environment in cHL and PTLD. In both of these lymphomas with known constitutive activation of AP-1, Gal1 expression is driven, in large part, by an AP-1–dependent Gal1 enhancer. The AP-1-dependent expression of Gal1 is a useful diagnostic marker and circulating Gal1 levels have prognostic value. In addition, neutralizing Gal1 antibodies have promising immunomodulatory activity in in vitro assays and tumor models. Engagement of PD-1 receptors triggers T-cell “exhaustion” and the progressive loss of effector T-cell function and proliferative capacity. In certain cancers, the tumor cell expression of PD-1 ligands inhibits T-cell activation and promotes the apoptosis of tumor-specific T cells. We recently integrated high-resolution copy number data with transcriptional profiles and identified the PD-1 ligands, PD-L1 and PD-L2, as key targets of the 9p24.1 amplification in cHL and a related lymphoid malignancy, primary mediastinal large B-cell lymphoma (MLBCL). PD-1 ligand gene amplification was associated with increased protein expression in both lymphoid malignancies. In cHLs and MLBCLs, the extended 9p24.1 amplification region also included the JAK2 locus. JAK2 amplification increased JAK2 protein expression and activity and specifically induced PD-1 ligand transcription. Chemical inhibition of JAK2 decreased PD-1 ligand expression. These findings defined 9p24.1 amplification as a disease-specific structural alteration that increased both the gene dosage of PD-1 ligands and their induction via JAK2. Like Gal1, PD-L1 includes an AP-1-dependent enhancer which augments PD-L1 expression in lymphomas with constitutive activation of AP-1. In addition, Epstein-Barr virus (EBV) induces PD-L1 expression via AP-1 and EBV+ cHLs with diploid 9p24.1 and the majority of EBV+ PTLDs express detectable PD-L1. Therefore, AP-1- and EBV-driven malignancies with normal 9p24.1 copy numbers, including cHLs and most PTLDs, may also be amenable to PD-1 blockade. Taken together, these studies identify the PD-1 axis and Gal1 as promising therapeutic targets in certain lymphoid malignancies and define genetic bases for increased expression of these immunoregulatory molecules. For these reasons, clinical evaluation of PD-1 blockade alone and in association with JAK2 inhibition is planned and assessment of antibody-mediated Gal1 blockade is underway. Citation Format: Margaret A. Shipp. Immune escape mechanisms in lymphoid malignancies. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr IA6.

First analysis of human herpesvirus 6T-cell responses: Specific boosting after HHV6 reactivation in stem cell transplantation recipients

Early human herpesvirus 6 (HHV6) reactivation after hematopoietic stem cell transplantation (HSCT) is associated with poor survival. We characterized HHV6 immuneresponses in HSCT patients during lymphopenia. Prospectively, HHV6 DNA-load was measured weekly by realtime-PCR. Numbers of IFNγ-producing HHV6-T-cells were retrospectively determined by enzyme-linked immunospot assay 2 months after HSCT. HHV6-specific T-cell proliferative capacity was analyzed with a newly developed assay using antigen-presenting autologous HHV6-infected PBMC. Fifty-six patients were included (median age 4.6 years; range 0.2-21.2 years). HHV6-reactivation occurred in 29/56 (52%) patients with a median time of 14 (range 1-41) days after HSCT. The median number of IFN-γ producing HHV6-specific T-cells at 2 months and the HHV6-specific CD8+ T-cell proliferative capacity at 6 months after HSCT was increased after HHV6-reactivation compared to non-reactivating patients (P=0.006 and p=0.019). In conclusion, HHV6-specific immuneresponses can be initiated during lymphopenia early after HSCT, which implicates a potential window for development of HHV6-specific (immuno)therapy.

CD8 T-Cell Proliferative Capacity Is Compromised in Primary HIV-1 Infection

Understanding the correlates of immunity that control HIV-1 infection is imperative to our understanding of HIV-1 disease and vaccine development. HIV-1-specific cytotoxic T lymphocytes are fundamental to the control of viremia; however, which T-cell repertoire components enact this control remains unclear. We hypothesize that polyfunctional HIV-1-specific CD8 T cells capable of viral control are present in most patients early in infection and these cells are distinguished by their ability to secrete interleukin (IL)-2 and proliferate. We examined HIV-1-specific CD8 T-cell proliferation and cytokine secretion in primary HIV-1 infection (PHI) using known HIV-1 cytotoxic T-cell epitopes to exclude CD4 bystander effect. We found that only a subset of patients with PHI demonstrated "CD4-independent" CD8 proliferation ex vivo. The remainder of the patients lacked HIV-1-specific CD8 T cells with proliferative capacity, even after the addition of exogenous IL-2. Among the proliferators, IL-2 production from the total HIV-specific CD8 T-cell population correlated with proliferation. Surprisingly, though, we did not routinely detect both IL-2 secretion and proliferative capacity from the same antigen-specific CD8 T cells. Thus, there are distinct and heterogeneous populations of CD8 T cells, phenotypically characterized by either proliferation or IL-2 secretion and few with dual capacity. Generation of these responses may be an important measure of HIV-1 control but are not universal after PHI. Furthermore, the heterogeneity of this population suggests that a simple measure of an effective vaccine response remains elusive.

Administration of anti-interleukin-6 monoclonal antibody prolongs cardiac allograft survival

To investigate the effects of anti-IL-6 monoclonal antibody (anti-IL-6 mAb) on acute allograft rejection and the potential mechanisms in a mouse heart transplantation model. Heterotopical heart graft model was performed. The anti-IL-6 mAb was administered to recipient mice after cardiac grafting. Results were compared with administration of anti-IL-17 mAb or anti-IL-6 mAb+anti-IL-17 mAb (the 'double' treatment). The cardiac allograft survival was monitored by daily palpation in combination with histological evaluation. Quantitative polymerase chain reaction assay, mixed lymphocyte reaction, and flow cytometric analysis were employed to determine the mRNA expression of pro-inflammatory cytokines, allogeneic T-cell proliferation, and the proportion of CD4(+) CD25(+) Foxp3(+) regulatory T cells in graft-infiltrating lymphocytes and splenocytes of recipients, respectively. The results showed that the cardiac allograft survival in anti-IL-6 mAb-treated mice was prolonged significantly when compared with that of the untreated or anti-IL-17 mAb-treated mice. Meanwhile, the 'double-treated' did not prolong graft survival significantly when compared with those treated with anti-IL-6 mAb. The increase of graft survival induced by anti-IL-6 mAb was associated with reduced transcript levels for IFN-γ and IL-17, accompanied by a dramatic reduction of T-cell proliferation capacity to alloantigen stimuli and a higher proportion of Treg cells. Thus, anti-IL-6 mAb may be protective against acute rejection after cardiac transplantation through suppressing the activation of effector T cells and promoting the induction of Treg cells.

No difference in Gag and Env immune-response profiles between vaccinated and non-vaccinated rhesus macaques that control immunodeficiency virus replication

Recent advances in human immunodeficiency virus (HIV) vaccine design have resulted in induction of strong CD4 T-cell proliferative and polyfunctional cytokine responses, which are also characteristic for long-term non-progressing (LTNP) HIV-infected individuals. However, limited information is available on the persistence of these responses after infection. Results from studies in non-human primates indicate that vaccine-induced immune responses are partially maintained upon viral infection and differ from the responses seen in non-vaccinated animals that typically progress to disease. However, it is unclear how these partially preserved responses compare to immune responses that are acquired naturally by LTNP animals. In this study, immune-response profiles were compared between vaccinated animals that, upon SHIV₈₉.₆ challenge, became infected but were able to control virus replication, and a group of animals having spontaneous control of this viral infection. Both groups were found to develop very similar immune responses with regard to induction of CD4 and CD8 T-cell polyfunctional cytokine responses, proliferative capacity and cytotoxic capacity, as measured by a standard ₅₁Cr release assay and more direct ex vivo and in vivo CTL assays. Hence, vaccinated animals that become infected, but control infection, appear to establish immune responses that are similar to those elicited by long-term non-progressors.

Inhibitory effects of progesterone differ in dendritic cells from female and male rodents

Steroid hormones, such as progesterone, are known to have immunomodulatory effects. Our research group previously reported direct effects of progesterone on dendritic cells (DCs) from female rodents. Primarily affecting mature DC function, progesterone effects included inhibition of proinflammatory cytokine secretion, downregulation of cell surface marker (major histocompatibility complex class II, CD80) expression, and decreased T-cell proliferative capacity, and were likely mediated through progesterone receptor (PR) because the PR antagonist RU486 reversed these effects. The goal of this study was to assess differences in response to progesterone by DCs from female and male rodents. Using real-time reverse-transcriptase polymerase chain reaction, transcriptional expression of steroid hormone receptors was measured in immature bone marrow-derived DCs (BMDCs) from male and female rats. Expression of steroid hormone receptor protein was also assessed in these cells using flow cytometry and fluorescence microscopy. To evaluate functional differences between BMDCs from female and male rats in response to the steroid hormone progesterone, levels of secreted cytokines were measured using enzyme-linked immunosorbent assay. Higher numbers of immature BMDCs from males expressed glucocorticoid receptor (GR) and androgen receptor (AR) proteins compared with females (males vs females, mean [SD]: GR = 68.75 [7.27] vs 43.61 [13.97], P = NS; AR = 75.99 [15.38] vs 8.25 [1.88], P = 0.002), whereas higher numbers of immature BMDCs from females expressed PR protein compared with males (females vs males: PR = 74.19 [12.11] vs 14.14 [4.55], P = 0.043). These differences were not found at the level of transcription (females vs males: GR = 0.088 vs 0.073, P = NS; AR = 0.076 vs 0.069, P = NS; PR = 0.075 vs 0.065, P = NS). Compared with those from females, mature BMDCs from males produced higher quantities of cytokines (tumor necrosis factor-alpha [TNF-alpha], interleukin [IL]-1beta, IL-10) (females vs males: TNF-alpha = 920.0 [79.25] vs 1100.61 [107.97], P = NS; IL-1beta = 146.60 [38.04] vs 191.10 [10.47], P = NS; IL-10 = 167.25 [4.50] vs 206.15 [23.48], P = NS). Conversely, BMDCs from females were more sensitive to progesterone, as indicated by a more dramatic reduction in proinflammatory cytokine secretion (females vs males, highest concentration of progesterone: TNF-alpha = 268.94 [28.59] vs 589.91 [100.98], P = 0.04; IL-1beta = 119.50 [10.32] vs 154.35 [6.22], P = NS). These findings suggest that progesterone effects on DCs in rodents may be more pronounced in females than in males, and this is likely due to differences in PR protein expression. Our observations may help elucidate disparities in the incidence and severity of autoimmune disorders between females and males, and the role specific steroid hormones play in regulating immune responses.

Selective Effects of Cyclosporine A on Th2-Skewed Dendritic Cells Matured With Viral-Like Stimulus By Means of Toll-Like Receptors

Successful prevention of allograft rejection and graft-versus-host disease with immunosuppression depends on controlled balance of Th1 and Th2 immune responses to establish tolerance and fight infection. Here, we have analyzed the effects of cyclosporine A (CsA) on the differentiation and functions of dendritic cells (DC2) that induce Th2 T cells. DC2 were differentiated from monocytes in the presence of CsA and were matured with viral or bacterial agonists (poly[I:C] or lipopolysaccharide). DC2 differentiation was not affected by CsA. In contrast, cytokine responses were altered with inhibition of interleukin-10 production in poly(I:C)-matured DC2. Surprisingly, interleukin-10 secretion by immature DC2 was increased after CsA treatment. Internalization was impaired in treated DC2, and CsA decreased the T-cell proliferative capacity of DC2 matured with poly(I:C), but not with lipopolysaccharide. In conclusion, CsA altered T-cell activating functions of DC2 with, notably, a regulatory phenotype for immature DC2 and opposite effects on poly(I:C)-matured cells.

Engineered Alt a 13 Fragment of Alternaria alternata Abrogated IgE Binding without Affecting T-cell Stimulation

Epitopes were delineated for allergenic proteins, but studies are required to identify residues mediating IgE binding. In the present study, the in silico approach was used to identify IgE-binding residues of Alt a 13(1-50) fragment and confirmed by experimental approach. IgE-binding epitopes of Alt a 13 mapped computationally were cloned, expressed, purified, and characterized using various immunochemical and biophysical methods. Among four fragments of Alt a 13, Alt a 13(1-50) demonstrated maximum IgE binding with two immunodominant regions and was mutated at these regions. The mutation in first region, Alt a 13(1-50)-K4A_S6F, did not show any change in immunological and biophysical properties of protein. However, mutations in the second region, Alt a 13(1-50)-T21F_N27I, caused reduced IgE binding, histamine release, and low IL-4 release on stimulation of Alternaria alternata positive patients peripheral blood mononuclear cells in vitro. This suggests that residues T21 and N27 are important for the secondary structure. In conclusion, Alt a 13(1-50)-T21F_N27I with reduced Th 2 response and intact T-cell proliferation capacity has potential for clinical use.

Predictive value of immune parameters before treatment interruption (TI) for CD4+T-cell count change during TI in HIV infection

BackgroundDespite the contraindications, stopping treatment for HIV infection continues to be a common practice. Understanding whether T-cell proliferative capacity and phenotypic markers before treatment interruption (TI) can predict CD4+T-cell count change and nadir during TI would be clinically useful.MethodsThis retrospective study included 27 HIV- infected patients in the chronic phase of infection while on combination antiretroviral therapy (cART) who underwent a TI. Peripheral blood mononuclear cells from a baseline pre-TI time point were screened for T-cell proliferation to cytomegalovirus (CMV) lysate, an HIV Gag p55 peptide pool as well as positive and negative control stimuli. CD28 and CD57 expression on CD4+and CD8+T-cells were measured.ResultsBaseline viral load, CD4+T-cell count, pre-cART nadir CD4+T-cell and percentage CD4+CD28+T-cells were all predictive of the lowest CD4+T-cell count during TI (Spearman's correlation P&lt;0.05 for all analyses). In addition, CD4+and CD8+T-cells proliferation to CMV lysate, baseline CD4+T-cell count and percentage CD4+CD57+T-cells correlated negatively with CD4+T-cell decrease during TI (Spearman's correlation P&lt;0.05 for all analyses).ConclusionsIn treated chronic HIV-infected patients, pre-TI immune parameters are potential predictors for both the nadir CD4+T-cell count and CD4+T-cell count decrease during TI.

Systemic CTLA-4 Blockade Ameliorates Glioma-Induced Changes to the CD4+ T Cell Compartment without Affecting Regulatory T-Cell Function

Patients with malignant glioma suffer global compromise of their cellular immunity, characterized by dramatic reductions in CD4(+) T cell numbers and function. We have previously shown that increased regulatory T cell (T(reg)) fractions in these patients explain T-cell functional deficits. Our murine glioma model recapitulates these findings. Here, we investigate the effects of systemic CTLA-4 blockade in this model. A monoclonal antibody (9H10) to CTLA-4 was employed against well-established glioma. Survival and risks for experimental allergic encephalomyelitis were assessed, as were CD4(+) T cell numbers and function in the peripheral blood, spleen, and cervical lymph nodes. The specific capacities for anti-CTLA-4 to modify the functions of regulatory versus CD4(+)CD25(-) responder T cells were evaluated. CTLA-4 blockade confers long-term survival in 80% of treated mice, without eliciting experimental allergic encephalomyelitis. Changes to the CD4 compartment were reversed, as anti-CTLA-4 reestablishes normal CD4 counts and abrogates increases in CD4(+)CD25(+)Foxp3(+)GITR(+) regulatory T cell fraction observed in tumor-bearing mice. CD4(+) T-cell proliferative capacity is restored and the cervical lymph node antitumor response is enhanced. Treatment benefits are bestowed exclusively on the CD4(+)CD25(-) T cell population and not T(regs), as CD4(+)CD25(-) T cells from treated mice show improved proliferative responses and resistance to T(reg)-mediated suppression, whereas T(regs) from the same mice remain anergic and exhibit no restriction of their suppressive capacity. CTLA-4 blockade is a rational means of reversing glioma-induced changes to the CD4 compartment and enhancing antitumor immunity. These benefits were attained through the conferment of resistance to T(reg)-mediated suppression, and not through direct effects on T(regs).

Long-Term Highly Active Antiretroviral Therapy in Chronic HIV-1 Infection: Evidence for Reconstitution of Antiviral Immunity

In this study we investigated the long-term effect of highly active antiretroviral therapy (HAART) on HIV-specific CD4+ T-cell responses in comparison with virus-specific CD4+ T-cell responses against the persistent herpes viruses cytomegalovirus (CMV) and Epstein-Barr virus (EBV). To this end, HIV- and herpes virus-specific cellular immune responses were measured longitudinally in 10 seroconverters with long-term follow-up including 55 months of successful suppression of viral load by HAART. HIV- and CMV-specific CD4+ T cells producing interferon-gamma (IFNgamma) or interleukin-2 (IL-2) were analysed as well as proliferative capacity. EBV-specific CD4+ T cells were determined using a 12-day ex vivo assay. Initiation of HAART resulted in a transient increase of HIV-specific IL-2(+)IFNgamma(+)CD4(+) T cells and, to a lesser extent, IL-2(+)CD4(+) T cells. Long-term HAART resulted in an increase in HIV-, CMV- and EBV-specific CD4+ T-cell proliferative capacity. The increase in HIV- and herpes-virus-specific CD4+ T-cell proliferative capacity after 55 months of HAART suggests that the improved proliferative response is not specific for HIV, but reflects a more general improvement of antiviral immune responses, which is induced by HAART.

The immunosuppressive effect of human cytomegalovirus infection in recipients of allogeneic hematopoietic stem cell transplantation

In immune-competent individuals, human cytomegalovirus (HCMV) infection is associated with impairment of T-cell function. Our goal was to evaluate prospectively whether clinically asymptomatic HCMV infection in allogeneic hematopoietic stem cell transplantation (alloHSCT) recipients, treated pre emptively with ganciclovir, influences T-cell function as well. Mitogen-stimulated T-cell proliferative activity, together with cell surface markers, was tested in 49 patients on days + 30, + 45, + 60, and + 90 after alloHSCT and, additionally, in cases of positive HCMV pp65-antigenemia. HCMV infection was diagnosed in 19 patients. None of them developed HCMV disease. T-cell proliferative activity was significantly decreased on days when HCMV antigenemia was positive as compared to days without antigenemia. The number of pp65-positive cells negatively correlated with proliferative response. Comparison of patients who did experience HCMV infection with those who did not reveals significant decrease of T-cell proliferative activity observed on days + 30 and + 45, a time period when antigenemia was most frequently found to be positive, whereas no difference was detected on days + 60 and + 90. We conclude that, even clinically asymptomatic, HCMV infection has negative impact on T-cell proliferation capacity in alloHSCT recipients. However, pre emptive therapy with ganciclovir makes this immunosuppressive effect transient and restricted to the time of infection duration.

Lymphocyte numbers and function in relation to periodontitis and smoking.

T and B lymphocytes play important roles in periodontitis. Smoking is considered a risk factor for periodontitis and may exert its negative effects through leukocytes. Taking smoking into consideration, the aim of this study was to analyze numbers of circulating T (CD3+) cells and their CD4+ and CD8+ subpopulations, B (CD19+) cells, and T-cell proliferative capacity in periodontitis. Lymphocyte immunophenotyping for T cells, their CD4+ and CD8+ subsets, and B cells was performed on peripheral blood from 76 periodontitis patients and 36 controls. Proliferative capacity of T cells was determined in whole-blood lymphocyte culture assays after mitogenic stimulation. Total T cells, CD4+ and CD8+ subpopulations, and responsiveness to specific T-cell stimuli did not differ between patients and controls; in addition, B cells were not significantly elevated in periodontitis patients. However, more periodontal breakdown in smoking patients was associated with higher numbers of CD3+ T cells, as well as with CD4+ and CD8+ T-cell subsets, and increased T-cell proliferation. Numbers of B cells were not affected by smoking. The increased numbers of T-cells and elevated T-cell responsiveness in patients who smoke may be one of several explanations why smoking is a risk factor for periodontitis. The mechanism of how T-cell function contributes to increase the severity of periodontal breakdown in smoking periodontitis patients needs to be investigated further.

IMMUNOMODULATORY EFFECTS OF GLASS FIBRES IN OCCUPATIONALLY EXPOSED WORKERS-COMPARISON WITH ASBESTOS

Background Recent research has implicated natural and man made mineral fibers as causes of lung disease. For these reasons, and because of the trend for substitution of asbestos with other man-made fibers, improved understanding, assessment and monitoring of the toxic effects of mineral fibers is increasingly important. Glass fibers belong to the family of synthetic inorganic fibers used as insulation materials. Methods Immune and hematological parameters were examined in 80 workers exposed to glass fibres and 36 in-factory controls; and 61 workers exposed to asbestos fibres and 46 town and 21 in-factory unexposed controls, respectively. Cellular immunity was determined by phenotypic analysis of human peripheral blood leukocytes (CD3, HLA DR, CD4, CD8, CD16+56, CD19). Immune function assays-proliferative response of lymphocytes and phagocytic activity of leukocytes were evaluated in exposed and unexposed populations. Humoral immunity was evaluated by levels of immunoglobulins and concentrations of interleukins in serum. Moreover expression of activation markers on eosinophils, expression of adhesion molecules on leukocytes and levels of soluble adhesion molecules in sera were assessed. Results Multifactorial analysis of variance showed significantly decreased expression of marker CD16+56 (natural killer cells) in people exposed to glass fibres. The proliferative capacity of T-cells was significantly altered in both populations of workers exposed to fibres. In the case of people from a former asbestos cement plant, significant suppression of T-cell proliferative activity was found. On the other hand significant elevation of T-cell proliferative capacity was found in workers exposed to glass fibres. The levels of soluble adhesion molecule ICAM-1 were higher in people from the asbestos plant. Moreover significantly increased expression of CD62L (L-selectin) on monocytes and granulocytes was found. Exposure to glass fibre increased levels of E-selectin in sera of exposed subjects. Significantly increased levels of immunoglobulin E, concentrations of IL-6 and IL-8 were found in abestos-exposed workers. Similar trend of elevated IL-8 was seen after glass fibre exposure. We found significantly increased expression in markers for activated eosinophils CD69 and CD66b among workers exposed to asbestos mineral fibres. Marker of CD66b was highly expressed also on peripheral blood eosinophils of humans exposed to glass fibres. Conclusions Our findings indicate that occupational exposure to both types of fibres modulates the immune response. Suppression of T-cell immunity and, to a lesser extent, B-cell immunity, were found in the case of exposure to asbestos, while stimulation of T-cell response was observed in the glass fibre exposed population. Increased levels of proinflammatory cytokines, levels of adhesion molecules in sera and elevated expression of activation markers on eosinophils may indicate hypersensitivity and an elevated inflammatory status in the exposed workers. This work was supported by the European Union (project no. QLK4-1999-01629, Fibretox), NIEHS Grant# 510 20 5240 00000 1 15010 9220 and Slovak Grant Agency for Science # 04.92.11.05.

Immunophenotyping and T-cell proliferative capacity in a healthy aged population.

The age-related decline of immunological functions is well established but it remains largely unknown which specific changes are related to disease. We analyzed peripheral blood lymphocytes of 42 healthy elderly as well as 24 healthy young subjects from southern Brazil. No differences in phytohemagglutinin-induced proliferation and CD4:CD8 ratio were found between the subjects. However, CD4 expression (considering mean fluorescence intensity) was found upregulated in elderly subjects. No changes in activation molecules CD25, CD28, CD69 and CD95 were observed. A reduced proportion of naive (CD45RA+) T cells was found in the elderly compared to young subjects. No changes in adhesion molecule expression (CD11c and CD31) were observed. However, the frequencies of CD49d-positive cells, as well as expression of CD62L, were increased in the eldery subjects. We further described two subgroups of eldery subjects with an immunological risk profile defined by lower CD4:CD8 ratio and reduced proliferative response to mitogens. These data suggest that healthy aging is associated with intact T-cell proliferation and some compensatory immunophenotypical changes.

Protective effect of vitamin E on lymphocyte growth capacity during incubation in vitro

Vitamin E, an essential lipid-soluble micronutrient, plays an important role in the immune system and serves as an immunostimulant in geriatric subjects. Using an in vitro incubation to simulate aging processes, we find that vitamin E mitigates loss of growth capacity in lymphocytes. Vitamin E presence during in vitro incubation did not affect significantly the level of peroxidation, the effects of exogenous IL-2, PGE 2, or indomethacin, nor levels of IL-2 production. Thus, the preservative effect on lymphocyte growth by vitamin E is not due primarily to its antioxidant function or to interleukin-2 or prostaglandin effects. The decreased growth capacity generated by in vitro incubation is accompanied by a variety of cellular alterations, including decreased CD5 surface antigen, enhanced suppression by adherent cells, and impaired communication between lymphocytes and adherent cells. The decrease in CD5 surface antigen correlates inversely with the cell density required for maximal cell proliferation, and the diminished CD5 levels were unaltered by vitamin E presence during the aging process. In contrast, protection of T-cell proliferative capacity by vitamin E in vitro correlates with diminished suppression by adherent cells and normalized interaction between lymphocytes and adherent cells.

Influence of elevated temperature on natural killer cell activity, lymphokine-activated killer cell activity and lectin-dependent cytotoxicity of human umbilical cord blood and adult blood cells

Purpose : To determine whether hyperthermia is to the benefit or detriment of host immune function, the effect of hyperthermia was evaluated on various functions of T-lymphocytes from human umbilical cord blood and compared to that of adult blood. Methods and Materials: Nonadherent mononuclear cells from cord blood or adult blood were used as the effector cells. To generate lymphokine activated killer (LAK) cells, effector cells were kept in culture for 5 days in complete medium containing recombinant human interleukin-2. To activate effector cells to become cytotoxic, cells were kept in culture in complete medium containing Con A. Cytotoxicity was determined in a standard 4-h chromium release assay using K-562 human erythroleukemic cells (in the natural killer cell activity assay) or Daudi cells (in the LAK cell activity or Lectin dependent cytotoxicity assay) as targets. For heat effects, cells in complete medium were heated at the desired temperature in a water bath for 1 h. Results: Lymphokine-activated killer cell activity, lectin-dependent cytotoxicity and T-cell proliferative capacity were not deficient in human cord blood. Cytotoxic activities of T-cells from adult blood as well as from cord blood can be enhanced at febrile range (⩽ 40°C), and were significantly decreased by exposure to 1 h at 42°C. Conclusion: The febrile responses (⩽ 40°C) to infection, in the course of malignant disease and with biological response modifiers treatment, may all be related to host defense mechanisms. Based on these observations, whole body hyperthermia (⩽ 40°C), in combination with the appropriate cytokines, may have therapeutic potential in the treatment of neonatal infections and malignancies under certain circumstances. Hyperthermia in febrile range may, therefore, confer an important immunoregulatory advantage to the host. In contrast, tumor killing therapeutic temperature (> 42°C) which inhibits host immunocompetence should probably be used only for local hyperthermia.