Use of recombinant ESAT-6:CFP-10 fusion protein for differentiation of infections of cattle by Mycobacterium bovis and by M. avium subsp. avium and M. avium subsp. paratuberculosis

Immunological diagnosis of Mycobacterium bovis infection of cattle is often confounded by cross-reactive responses resulting from exposure to other mycobacterial species, especially Mycobacterium avium. Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are dominant gamma interferon (IFN-gamma)-inducing antigens of tuberculous mycobacteria, and they are absent from many environmental nontuberculous mycobacteria. Because M. avium exposure is the primary confounding factor in the diagnosis of M. bovis-infected animals, in vitro responses to a recombinant ESAT-6:CFP-10 (rESAT-6:CFP-10) fusion protein by blood leukocytes from cattle naturally exposed to M. avium or experimentally challenged with Mycobacterium avium subsp. avium or Mycobacterium avium subsp. paratuberculosis were compared to responses by M. bovis-infected cattle. Responses to heterogeneous mycobacterial antigens (i.e., purified protein derivatives [PPDs] and whole-cell sonicates [WCSs]) were also evaluated. Tumor necrosis factor alpha (TNF-alpha), IFN-gamma, and nitric oxide responses by M. bovis-infected cattle to rESAT-6:CFP-10 exceeded (P < 0.05) the corresponding responses by cattle naturally sensitized to M. avium. Experimental infection with M. bovis, M. avium, or M. avium subsp. paratuberculosis induced significant (P < 0.05) IFN-gamma and nitric oxide production to WCS and PPD antigens, regardless of the mycobacterial species used for the preparation of the antigen. Responses to homologous crude antigens generally exceeded responses to heterologous antigens. Nitric oxide and IFN-gamma responses to rESAT-6:CFP-10 by blood leukocytes from M. bovis-infected calves exceeded (P < 0.05) the corresponding responses of noninfected, M. avium-infected, and M. avium subsp. paratuberculosis-infected calves. Despite the reported potential for secretion of immunogenic ESAT-6 and CFP-10 proteins by M. avium and M. avium subsp. paratuberculosis, it appears that use of the rESAT-6:CFP-10 fusion protein will be useful for the detection of tuberculous cattle in herds with pre-existing sensitization to M. avium and/or M. avium subsp. paratuberculosis.     

Antigenic equivalence of human T-cell responses to Mycobacterium tuberculosis-specific RD1-encoded protein antigens ESAT-6 and culture filtrate protein 10 and to mixtures of synthetic peptides

The early secreted antigenic target 6-kDa protein (ESAT-6) and culture filtrate protein 10 (CFP-10) are promising antigens for reliable immunodiagnosis of tuberculosis. Both antigens are encoded by RD1, a genomic region present in all strains of Mycobacterium tuberculosis and M. bovis but lacking in all M. bovis bacillus Calmette-Guérin vaccine strains. Production and purification of recombinant antigens are laborious and costly, precluding rapid and large-scale testing. Aiming to develop alternative diagnostic reagents, we have investigated whether recombinant ESAT-6 (rESAT-6) and recombinant CFP-10 (rCFP-10) can be replaced with corresponding mixtures of overlapping peptides spanning the complete amino acid sequence of each antigen. Proliferation of M. tuberculosis-specific human T-cell lines in response to rESAT-6 and rCFP-10 and that in response to the corresponding peptide mixtures were almost completely correlated (r = 0.96, P < 0.0001 for ESAT-6; r = 0.98, P < 0.0001 for CFP-10). More importantly, the same was found when gamma interferon production by peripheral blood mononuclear cells in response to these stimuli was analyzed (r = 0.89, P < 0.0001 for ESAT-6; r = 0.89, P < 0.0001 for CFP-10). Whole protein antigens and the peptide mixtures resulted in identical sensitivity and specificity for detection of infection with M. tuberculosis. The peptides in each mixture contributing to the overall response varied between individuals with different HLA-DR types. Interestingly, responses to CFP-10 were significantly higher in the presence of HLA-DR15, which is the major subtype of DR2. These results show that mixtures of synthetic overlapping peptides have potency equivalent to that of whole ESAT-6 and CFP-10 for sensitive and specific detection of infection with M. tuberculosis, and peptides have the advantage of faster production at lower cost.     

Bacterial protein secretion is required for priming of CD8+ T cells specific for the Mycobacterium tuberculosis antigen CFP10

Mycobacterium tuberculosis infection elicits antigen-specific CD8(+) T cells that are required to control disease. It is unknown how the major histocompatibility complex class I (MHC-I) pathway samples mycobacterial antigens. CFP10 and ESAT6 are important virulence factors secreted by M. tuberculosis, and they are immunodominant targets of the human and murine T-cell response. Here, we test the hypothesis that CFP10 secretion by M. tuberculosis is required for the priming of CD8(+) T cells in vivo. Our results reveal an explicit dependence upon the bacterial secretion of the CFP10 antigen for the induction of antigen-specific CD8(+) T cells in vivo. By using well-defined M. tuberculosis mutants and carefully controlling for virulence, we show that ESX-1 function is required for the priming of CD8(+) T cells specific for CFP10. CD4(+) and CD8(+) T-cell responses to mycobacterial antigens secreted independently of ESX-1 were unaffected, suggesting that ESX-1-dependent phagosomal escape is not required for CD8(+) T-cell priming during infection. We propose that the overrepresentation of secreted proteins as dominant targets of the CD8(+) T-cell response during M. tuberculosis infection is a consequence of their preferential sampling by the MHC-I pathway. The implications of these findings should be considered in all models of antigen presentation during M. tuberculosis infection and in vaccine development.     

Mycobacterial ESAT-6 protein enhances mouse IFN-gamma responses to Mycoplasma hyopneumoniae P71 protein.

Type 1 immune responses play an important role in the resolution of diseases with infectious or oncogenic etiologies. Vaccines for production animals frequently target humoral immune responses and are often ineffective in protecting against disease. In order to shift the immune response more toward cellular immunity (i.e., type 1 response), we tested the ability of a mycobacterial protein, early secretory antigenic target (ESAT-6), to enhance interferon-gamma (IFN-gamma) secretion during the recall response with a second antigen. The Mycoplasma hyopneumoniae membrane protein P71 was used as a test antigen in murine vaccination studies. The ESAT-6 open reading frame (ORF) was fused to DNA encoding P71 to produce a recombinant protein that was used to immunize BALB/c mice. Control mice immunized with P71 alone demonstrated a splenic response characterized by release of interleukin-10 (IL-10) and a balanced antigen-specific IgG1/IgG2a antibody response. The presence of ESAT-6 as a fusion partner with P71 during immunization, however, resulted in an enhanced P71-specific IFN-gamma response, decreased release of IL-10, and significantly greater (p < 0.05) IgG2a antibody levels in comparison to immunizing with P71 alone. These results demonstrate that ESAT-6 can modify the profile of an immunologic response to an accompanying immunogen.     

Use of Recombinant ESAT-6:CFP-10 Fusion Protein for Differentiation of Infections of Cattle by Mycobacterium bovis and by M. avium subsp. avium and M. avium subsp. paratuberculosis

Immunological diagnosis of Mycobacterium bovis infection of cattle is often confounded by cross-reactive responses resulting from exposure to other mycobacterial species, especially Mycobacterium avium. Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are dominant gamma interferon (IFN-γ)-inducing antigens of tuberculous mycobacteria, and they are absent from many environmental nontuberculous mycobacteria. Because M. avium exposure is the primary confounding factor in the diagnosis of M. bovis-infected animals, in vitro responses to a recombinant ESAT-6:CFP-10 (rESAT-6:CFP-10) fusion protein by blood leukocytes from cattle naturally exposed to M. avium or experimentally challenged with Mycobacterium avium subsp. avium or Mycobacterium avium subsp. paratuberculosis were compared to responses by M. bovis-infected cattle. Responses to heterogeneous mycobacterial antigens (i.e., purified protein derivatives [PPDs] and whole-cell sonicates [WCSs]) were also evaluated. Tumor necrosis factor alpha (TNF-α), IFN-γ, and nitric oxide responses by M. bovis-infected cattle to rESAT-6:CFP-10 exceeded (P < 0.05) the corresponding responses by cattle naturally sensitized to M. avium. Experimental infection with M. bovis, M. avium, or M. avium subsp. paratuberculosis induced significant (P < 0.05) IFN-γ and nitric oxide production to WCS and PPD antigens, regardless of the mycobacterial species used for the preparation of the antigen. Responses to homologous crude antigens generally exceeded responses to heterologous antigens. Nitric oxide and IFN-γ responses to rESAT-6:CFP-10 by blood leukocytes from M. bovis-infected calves exceeded (P < 0.05) the corresponding responses of noninfected, M. avium-infected, and M. avium subsp. paratuberculosis-infected calves. Despite the reported potential for secretion of immunogenic ESAT-6 and CFP-10 proteins by M. avium and M. avium subsp. paratuberculosis, it appears that use of the rESAT-6:CFP-10 fusion protein will be useful for the detection of tuberculous cattle in herds with pre-existing sensitization to M. avium and/or M. avium subsp. paratuberculosis.     

Multiple Mycobacterium antigens induce interferon-gamma production from sarcoidosis peripheral blood mononuclear cells

Studies of sarcoidosis immunology have noted oligoclonal T cell populations, suggesting cell-mediated immunity that is antigen-specific. Sarcoidosis immunology and pathology are most similar to mycobacterial infections. Mycobacterium tuberculosis infection in mice and humans reflects T helper 1 (Th1) immune responses to multiple cell wall and secreted antigens. We investigated if the oligoclonal immune response in individual sarcoidosis subjects could be elicited by multiple secreted mycobacterial antigens by performing ex vivo enzyme-linked immunospot assay (ELISPOT) on peripheral blood mononuclear cells (PBMC) from 30 sarcoidosis, 26 purified protein derivative negative (PPD-) control and 10 latent tuberculosis subjects (PPD+) to assess Th1 responses to mycobacterial superoxide dismutase A (sodA), catalase-peroxidase (katG) and early secreted antigenic target protein (ESAT-6). A significant difference was noted among the sarcoidosis and PPD- control subjects to ESAT-6 [12 of 30 versus one of 26 (P = 0.0014)], katG [nine of 30 versus none of 26 (P = 0.002)] and sodA [12 of 30 versus none of 26 (P = 0.002)]. There was no significant difference between sarcoidosis and PPD+ subjects. Twelve sarcoidosis subjects recognized two or more mycobacterial proteins, as well as multiple distinct epitopes within individual proteins. One sarcoidosis subject on whom we collected bronchoalveolar lavage (BAL) fluid and PBMC had no recognition of mycobacterial antigens using PBMC, but BAL fluid demonstrated strong Th1 immune responses to ESAT-6 and katG. Individual sarcoidosis subjects recognized not only multiple mycobacterial proteins, but multiple distinct peptides within a specific protein, thus demonstrating that multiple mycobacterial epitopes elicit the Th1 immune response observed. Immune responses by sarcoidosis T cells to mycobacterial proteins may have an important role in sarcoidosis pathogenesis.     

Cattle husbandry in Ethiopia is a predominant factor affecting the pathology of bovine tuberculosis and gamma interferon responses to mycobacterial antigens

Bovine tuberculosis is a major economic problem and a potential public health risk. Improved diagnostics like the gamma interferon (IFN-gamma) test with ESAT6 and/or CFP10 could contribute to the control program. We assessed IFN-gamma responses in zebu (Ethiopian Arsi breed) and Holstein cattle kept indoors or in a pasture to tuberculin purified protein derivative (PPD) and an ESAT6-CFP10 protein cocktail. Furthermore, the intensity and distribution of pathology of bovine tuberculosis were compared between the two breeds. Our data demonstrated significantly (all P < 0.02) higher IFN-gamma responses to avian PPD, bovine PPD, and the ESAT6-CFP10 protein cocktail in Holstein than in zebu cattle, while lesion severities in infected animals and tuberculin skin test responses did not differ significantly (P > 0.05) between the two breeds. Holstein cattle that were kept indoors produced significantly (all P < 0.01) higher IFN-gamma levels in response to avian PPD, bovine PPD, and the ESAT6-CFP10 protein cocktail than did Holstein cattle kept in a pasture. Moreover, lesion severity was significantly higher in Holstein cattle kept indoors (P = 0.001) than in those kept in the pasture. Lesions were localized predominantly in the digestive tract in cattle kept in a pasture, while they were localized in the respiratory tract in cattle kept indoors. In conclusion, in Holstein cattle, husbandry was a dominant factor influencing the severity of tuberculosis lesions and IFN-gamma responses to mycobacterial antigens compared to breed. A difference in the cellular immune response between zebu and Holstein cattle was observed, while tuberculosis lesion severities were identical in the two breeds, when both were kept in a pasture.     

Activated T lymphocytes in pre-eclampsia

PROBLEM: The aim of our study was to investigate the activation markets of T CD3(+), T helper CD4(+) and T cytotoxic CD8(+) cells, as well as, the populations of T na?ve CD4(+) CD45RA(+), T memory CD4(+) CD45RO(+) and T regulatory lymphocytes in PE and healthy pregnant women. METHOD OF STUDY: Twenty-five patients with PE and thirty healthy third trimester pregnant women were included in the study. Peripheral blood mononuclear cells were isolated from peripheral blood, stained with monoclonal antibodies and estimated using the flow cytometric method. RESULTS: The percentages of CD4(+)CD25(+), CD4(+)CD25(dim), CD3(+)HLA-DR(+), CD4(+)HLA-DR(+) and CD8(+)HLA-DR(+) cells did not differ between study groups. The population of T regulatory CD4(+)CD25(bright) lymphocytes was significantly lower in the group of patients with PE when compared with the controls (P < 0.01). The percentages of CD3(+)CD25(+) (P < 0.05), CD8(+)CD25(+) (P < 0.05), CD4(+)45RO(+) (P < 0.01) lymphocytes were significantly higher, while CD4(+)CD45RA(+) (P < 0.01) cells--significantly lower in peripheral blood of patients with PE when compared with the control group. CONCLUSION: The increased levels of T CD4(+)45RO(+) and T CD8(+) CD25(+) cells can suggest the activation of CD4(+) and CD8(+) T lymphocytes in pre-eclampsia. It seems possible that the activation of T lymphocytes is associated with the deficiency of T regulatory cells in PE.     

HIV-infected children with moderate/severe immune-suppression: changes in the immune system after highly active antiretroviral therapy

The objective of this study was to monitor the changes in the immune system of HIV-infected children with moderate or severe immunodeficiency after highly active antiretroviral therapy (HAART), comprising a follow-up study in 14 HIV-infected children on HAART at two time points separated approximately by 11.8 +/- 0.4 (9.9; 15.4) months. HIV-infected children had significantly lower TREC levels than the control group, but 1 year after HAART the levels increased significantly (P < 0.05). In contrast, viral load (VL) did not change significantly. A positive correlation between T cell receptor excision circle (TREC) levels and both CD4(+) T cell absolute counts (r = 0.558; P = 0.05) and percentages (r = 0.625; P = 0.030) was found. During follow-up on HAART, the percentages and absolute counts of naive CD4(+) and CD8(+) T cell subsets were increased significantly (P < 0.05). CD4(+) CD45RA(hi+) CD62L(+), CD4(+) CD45RA(+) and CD4(+) CD38(+) percentages, and the CD8(+) CD45RA(hi+) CD62L(+) counts reached similar values to the control group. Also, CD8(+) CD45RO(+) CD38(+) and CD8(+) CD45RO(+) percentages, and CD8(+) CD45RO(+) CD38(+) absolute counts (P < 0.05) decreased with respect to the baseline. Lymphoproliferative responses to pokeweed mitogen (PWM) before HAART were lower in HIV-infected children than the control group, but they recovered to normal levels after a year on HAART. Tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma production by PHA-activated peripheral blood mononuclear cells (PBMC) was lower before HAART (P < 0.001), but reached similar levels to the control group 1 year after HAART. In HIV-infected children IgG, IgG(1) and IgG(3) plasma levels decreased significantly after HAART. The immune system reconstitution induced by HAART in HIV-infected children seems to be the consequence of decreased immune system activation and naive T cell reconstitution, mainly of thymic origin.     

Recognition of mycobacterial antigens delivered by genetically detoxified Bordetella pertussis adenylate cyclase by T cells from cattle with bovine tuberculosis

The exponential increase in the incidence of tuberculosis in cattle over the last two decades in the British national herd constitutes a significant economic problem. Therefore, research efforts are under way to develop cattle tuberculosis vaccines and specific diagnostic reagents to allow the distinction of vaccinated from infected animals. Mycobacterial antigens like ESAT-6 and CFP10 allow this distinction. This study investigates whether fusion protein of ESAT-6 or CFP10 with genetically detoxified Bordetella pertussis adenylate cyclase (CyaA) are recognized by Mycobacterium bovis-infected cattle more effectively than conventional recombinant proteins are, thus enhancing sensitivity or reducing the amount of antigens required. By measuring the frequencies of gamma interferon (IFN-gamma)-producing cells, we were able to show that the presentation of CFP10 as a CyaA fusion protein enhanced the molecular efficiency of its recognition 20-fold, while the recognition of ESAT-6 was not improved by CyaA delivery. Furthermore, in the whole-blood IFN-gamma test currently used in the field, the delivery of CFP10 and ESAT-6 by fusion to CyaA increased the amount of IFN-gamma produced and hence the proportion of infected animals responding to CFP10. The improved T-cell recognition of CyaA336/CFP10 was found to be dependent upon interaction with CD11b. In addition, presentation of CyaA336/CFP10 to CD4+ T cells was chloroquine sensitive, and CFP10 delivery by CyaA resulted in its accelerated presentation to T cells. In conclusion, the use of CyaA fusion proteins with ESAT-6 and CFP10 has the potential to improve the sensitivity of immunodiagnostic tests detecting bovine tuberculosis in cattle.     

Evaluation of thymopoiesis using T cell receptor excision circles (TRECs): differential correlation between adult and pediatric TRECs and naïve phenotypes

To determine whether the thymus is still functional despite age-related involution, we measured a biomarker for thymopoiesis known as the T cell receptor excision circle (TREC) from peripheral blood mononuclear cells (PBMCs) of 148 healthy children and from PBMCs, CD4(+), and CD8(+) cells of 32, 30, and 50 healthy adults, respectively. We demonstrate that during the first 5 years of life, thymic output is decreased (P 0.002) but not dramatically (r = -0. 282). Among adults aged 23-58, thymic output was inversely correlated with age, as measured from PBMCs (r = -0.628, P < 0.0005), CD4(+) (r = -0.530, P 0.003), and CD8(+) fractions (r = -0.385, P 0. 006). A strong correlation existed between pediatric PBMC TRECs and the expression of three na?ve phenotypic markers (CD45RA(+)CD45RO(-), CD45RA(+)CD62L(+), and CD45RO(-)CD27(+)CD95(low)). Adult PBMC TRECs correlated only with the expression of CD45RA(+)CD45RO(-) (r = 0.459, P 0.012). Our data suggest that in adults CD45RA(+)CD45RO(-) may be enriched for TRECs and add to a growing body of evidence illustrating intact thymic function in adulthood.     

Responses of bovine WC1(+) gammadelta T cells to protein and nonprotein antigens of Mycobacterium bovis

WC1(+) gammadelta T cells of Mycobacterium bovis-infected cattle are highly responsive to M. bovis sonic extract (MBSE). In mycobacterial infections of other species, gammadelta T cells have been shown to respond to protein and nonprotein antigens, but the bovine WC1(+) gammadelta T-cell antigenic targets within MBSE require further definition in terms of the dominance of protein versus nonprotein components. The present study sought to characterize the WC1(+) gammadelta T-cell antigenic targets, together with the role of interleukin-2 (IL-2), in the context of M. bovis infection. This was achieved by testing crude and defined antigens to assess protein versus nonprotein recognition by WC1(+) gammadelta T cells in comparison with CD4(+) alphabeta T cells. Both cell types proliferated strongly in response to MBSE, with CD4(+) T cells being the major producers of gamma interferon (IFN-gamma). However, enzymatic digestion of the protein in MBSE removed its ability to stimulate CD4(+) T-cell responses, whereas some WC1(+) gammadelta T-cell proliferation remained. The most antigenic protein inducing proliferation and IFN-gamma secretion in WC1(+) gammadelta T-cell cultures was found to be ESAT-6, which is a potential novel diagnostic reagent and vaccine candidate. In addition, WC1(+) gammadelta T-cell proliferation was observed in response to stimulation with prenyl pyrophosphate antigens (isopentenyl pyrophosphate and monomethyl phosphate). High levels of cellular activation (CD25 expression) resulted from MBSE stimulation of WC1(+) gammadelta T cells from infected animals. A similar degree of activation was induced by IL-2 alone, but for WC1(+) gammadelta T-cell division IL-2 was found to act only as a costimulatory signal, enhancing antigen-driven responses. Overall, the data indicate that protein antigens are important stimulators of WC1(+) gammadelta T-cell proliferation and IFN-gamma secretion in M. bovis infection, with nonprotein antigens inducing significant proliferation. These findings have important implications for diagnostic and vaccine development.     

Tumour-infiltrating T-cell subsets, molecular changes in colorectal cancer, and prognosis: cohort study and literature review

The abundance of tumour-infiltrating T-cells has been associated with microsatellite instability (MSI) and a favourable prognosis in colorectal cancer. However, numerous molecular alterations have been associated with clinical outcome, and potentially confounding the biological and prognostic significance of tumour-infiltrating T-cells. We utilized a database of clinically and molecularly-annotated colon and rectal carcinoma cases (N = 768; stage I-IV) in two prospective cohort studies (the Nurses' Health Study and the Health Professionals Follow-up Study) and quantified the densities of CD3(+), CD8(+), CD45RO(+) (PTPRC), and FOXP3(+) cells within neoplastic epithelial areas using an Ariol image analysis system and tissue microarray. We used Cox proportional hazard models to compute the mortality hazard ratio, adjusting for clinical and molecular features including KRAS, BRAF, and PIK3CA mutations, MSI, CIMP, and LINE-1 hypomethylation. The densities of CD8(+), CD45RO(+), and FOXP3(+) cells were significantly associated with patient survival in univariate analyses (P(trend) < 0.007). In the multivariate model, tumour-infiltrating CD45RO(+)-cell density, but not CD3(+), CD8(+) or FOXP3(+)-cell density, was significantly associated with survival (p = 0.0032). In multivariate linear regression analysis, MSI-high (p < 0.0001) and high-level tumour LINE-1 methylation (p = 0.0013) were independently associated with higher CD45RO(+)-cell density. The survival benefit associated with CD45RO(+) cells was independent of MSI and LINE-1 status. In conclusion, tumour-infiltrating CD45RO(+)-cell density is a prognostic biomarker associated with longer survival of colorectal cancer patients, independent of clinical, pathological, and molecular features. In addition, MSI-high and tumour LINE-1 methylation level are independent predictors of CD45RO(+)-cell density. Our data offer a possible mechanism by which MSI confers an improved clinical outcome and support efforts to augment the host immune response in the tumour microenvironment as a strategy of targeted immunotherapy.     

MHC class II tetramer guided detection of Mycobacterium tuberculosis-specific CD4+ T cells in peripheral blood from patients with pulmonary tuberculosis

Novel diagnostic tools are needed to diagnose latent infection and to provide biologically meaningful surrogate markers to define cellular immune responses against Mycobacterium tuberculosis (MTB). Interferon gamma-based assays have recently been developed in addition to the more than 100-year-old tuberculin skin test (TST) for the immune diagnosis of MTB in blood. The advent of soluble MHC/peptide tetramer molecules allows to objectively enumerate antigen-specific T cells. We identified novel MHC class II-restricted MTB epitopes and used HLA-DR4 tetrameric complexes to visualize ex vivo CD4(+) T cells directed against the antigens Ag85B and the 19-kDa lipoprotein, shared between MTB and other Mycobacterium species, and CD4(+) T cells which recognize the MTB-associated ESAT-6 antigen. MTB-reactive CD4(+) T cells reside predominantly in the CD45RA(+) CD28(+) and CD45(-) CD28(+) T-cell subset and recognize naturally processed and presented MTB epitopes. HLA-DR4-restricted, Ag85B or ESAT-6-specific CD4(+) T cells show similar dynamics over time in peripheral blood mononuclear cells (PBMC) when compared with CD8(+) T cells directed against the corresponding HLA-A2-presented MTB epitopes in patients with pulmonary MTB infection and subsequent successful therapy. This was not found to be true for T-cell responses directed against the 19-kDa lipoprotein. The dissection of the cellular immune response in M. tuberculosis infection will enable novel strategies for monitoring MTB vaccine candidates and to gauge CD4(+) T cells directed against MTB.     

Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens

Culture filtrate from Mycobacterium tuberculosis contains protective antigens of relevance for the generation of a new antituberculosis vaccine. We have identified two previously uncharacterized M. tuberculosis proteins (TB7.3 and TB10.4) from the highly active low-mass fraction of culture filtrate. The molecules were characterized, mapped in a two-dimensional electrophoresis reference map of short-term culture filtrate, and compared with another recently identified low-mass protein, CFP10 (F. X. Berthet, P. B. Rasmussen, I. Rosenkrands, P. Andersen, and B. Gicquel. Microbiology 144:3195-3203, 1998), and the well-described ESAT-6 antigen. Genetic analyses demonstrated that TB10.4 as well as CFP10 belongs to the ESAT-6 family of low-mass proteins, whereas TB7.3 is a low-molecular-mass protein outside this family. The proteins were expressed in Escherichia coli, and their immunogenicity was tested in cultures of peripheral blood mononuclear cells from human tuberculosis (TB) patients, Mycobacterium bovis BCG-vaccinated donors, and nonvaccinated donors. The two ESAT-6 family members, TB10.4 and CFP10, were very strongly recognized and induced gamma interferon release at the same level (CFP10) as or at an even higher level (TB10.4) than ESAT-6. The non-ESAT-6 family member, TB7.3, for comparison, was recognized at a much lower level. CFP10 was found to distinguish TB patients from BCG-vaccinated donors and is, together with ESAT-6, an interesting candidate for the diagnosis of TB. The striking immunodominance of antigens within the ESAT-6 family is discussed, and hypotheses are presented to explain this targeting of the immune response during TB infection.     

Cytokine expression in the lower airways of nonasthmatic subjects with allergic rhinitis: influence of natural allergen exposure

BACKGROUND: Natural exposure to pollen provokes an increase in airway responsiveness in nonasthmatic subjects with seasonal allergic rhinitis. This natural exposure may induce inflammatory cell recruitment and cytokine release, leading to lower airway inflammation. OBJECTIVE: The aim of this study was to characterize lower airway inflammation in nonasthmatic pollen-sensitive subjects. METHODS: We performed immunohistochemical tests on bronchial biopsy specimens from subjects with rhinitis who had no past or current history of asthma to evaluate cytokine expression and inflammatory cell numbers and activation both in and out of the pollen season. RESULTS: The number of CD4(+), CD8(+), and CD45RO(+) lymphocyte subpopulations were significantly higher during the pollen season compared with the out-of-season period (P <.04). Furthermore, EG1(+) cells tended to increase after natural pollen exposure (P =.06). The number of IL-5(+) cells increased significantly after natural exposure to pollen compared with out-of-season numbers (P <.01). This increase in IL-5 expression was correlated with the numbers of CD3(+), CD4(+), CD45RO(+), and EG1(+) cells. The numbers of tryptase-positive, IFN-gamma(+), and IL-4(+) cells did not change after natural exposure. CONCLUSION: This study showed that natural pollen exposure was associated with an increase in lymphocyte numbers, eosinophil recruitment, and IL-5 expression in the bronchial mucosa of nonasthmatic subjects with allergic rhinitis.     

Primary and secondary human in vitro T-cell responses to soluble antigens are mediated by subsets bearing different CD45 isoforms.

A culture system has been developed which consistently supports in vitro proliferative responses to conventional soluble antigens by human CD4+ T cells from non-immunized donors. T cells exposed to an antigen in primary cultures could be restimulated in vitro in an antigen-specific manner to give secondary responses with greater magnitudes and a more rapid onset than the initial reaction. To characterize further the responding T-cell population in primary compared with secondary reactions, T cells were depleted of CD45RA+ or CD45RO+ cells and stimulated with recall and non-recall antigens. It was found that the soluble non-recall antigen keyhole limpet haemocyanin did not stimulate CD45RO+ T cells, yet induced strong proliferative responses from CD45RA+ T cells. Conversely, it was confirmed that human CD45RO+ T cells respond to the recall antigen-purified protein derivative from Mycobacterium tuberculosis. Cell mixing experiments indicated that CD45RO+ T cells are unlikely to have any suppressive effect on the reactivity of CD45RA+ cells to non-recall antigens. These data provide new support for the hypothesis that CD45RA+ represents the naive and CD45RO+ the memory phenotype of human CD4+ T cells.     

Pre-eclampsia affects the immunophenotype of neonates

OBJECTIVE: We tried to estimate whether immunological changes are present in neonates born to mothers who had been suffering from pre-eclampsia. STUDY DESIGN: Eighteen neonates born to mothers with severe pre-eclampsia (between 35 and 40 weeks of gestation) and 20 full-term healthy newborns (between 38 and 40 weeks of gestation) were included in the study. The lymphocytes were isolated from umbilical cord blood. The specific lymphocyte antigens were determined using direct staining with monoclonal antibodies and analysed by flow-cytometry. RESULTS: We observed that neonates born to pre-eclamptic mothers had decreased percentage of T CD 3(+), CD 4(+) and T CD 8(+)28(+) (cytotoxic) lymphocytes and increased percentage CD 3(-)16/56(+) cells and CD 8(+)28(-) (suppressor) lymphocytes in comparison with newborns of healthy women. Furthermore, we found decreased CD 4: CD 8 lymphocyte ratio in the study group in comparison with the control group. We also observed that the percentage of CD 19(+)5(+), CD 4(+)8(+), CD 19(+)40(+) and CD 3(+)40L(+) lymphocytes did not differ in both studied groups. The percentage of CD 4(+)45RO(+), CD 8(+)45RO(+) memory cells was higher in neonates born to pre-eclamptic mothers when compared to controls. Moreover, the expression of CD 25 molecule was higher on T CD 8(+) and B CD 19(+) lymphocytes of neonates of pre-eclamptic mothers. CONCLUSION: The alterations in the immunological parameters of neonates born to pre-eclamptic mothers can be associated with the maternal disease.     

Levels of expression of complement regulatory proteins CD46, CD55 and CD59 on resting and activated human peripheral blood leucocytes

The cell surface complement regulatory (CReg) proteins CD46, CD55 and CD59 are widely expressed on human lymphoid and non-lymphoid cells. This study aimed to compare systematically levels of CReg expression by different leucocyte subsets and to determine whether levels were increased following activation in vitro. Levels of each CReg protein were similar on freshly isolated monocytes and all major lymphocyte subsets, except that CD4(+) cells expressed significantly less CD46 than CD8(+) cells (P < 0.05) while the reverse was observed for CD55 (P < 0.02). CD56(+) cells, predominantly natural killer cells, expressed significantly lower levels of CD59 than T cells (P < 0.02). CD45RO(+) cells had higher levels of surface CD46 and CD59, but lower levels of CD55, than CD45RO(-) cells (P < 0.02); CD25(+) cells also expressed significantly less CD55 than CD25(-) cells (P < 0.002). Neutrophils expressed higher levels of CD59, but lower levels of CD55, than monocytes. Following activation with phytohaemagglutinin, CD46 was up-regulated on all leucocyte subsets with the exception of CD56(+) cells. Both CD55 and CD59 were also markedly up-regulated on monocytes, and CD55 expression was greater on CD8(+) than CD4(+) cells following activation (P < 0.02). Lipopolysaccharide treatment did not significantly alter B-cell expression of CReg proteins whereas CD55 and CD59, but not CD46, were significantly up-regulated on monocytes (P < 0.02). These observations that CReg proteins are up-regulated on certain activated leucocyte subsets indicate that levels would be increased following immune responses in vivo. This could enhance both protection against local complement activation at inflammatory sites and also the immunoregulatory properties of these leucocytes.