Exposure to Mycobacterium avium can modulate established immunity against Mycobacterium tuberculosis infection generated by Mycobacterium bovis BCG vaccination

Mycobacterium bovis bacille Calmette Guerin (BCG), the current vaccine against infection with Mycobacterium tuberculosis, offers a variable, protective efficacy in man. It has been suggested that exposure to environmental mycobacteria can interfere with the generation of BCG-specific immunity. We hypothesized that exposure to environmental mycobacteria following BCG vaccination would interfere with established BCG immunity and reduce protective efficacy, thus modeling the guidelines for BCG vaccination within the first year of life. Mice were vaccinated with BCG and subsequently given repeated oral doses of live Mycobacterium avium to model exposure to environmental mycobacteria. The protective efficacy of BCG with and without subsequent exposure to M. avium was determined following an aerogenic challenge with M. tuberculosis. Exposure of BCG-vaccinated mice to M. avium led to a persistent increase in the number of activated T cells within the brachial lymph nodes but similar T cell activation profiles in the lungs following infection with M. tuberculosis. The capacity of BCG-vaccinated mice to reduce the bacterial load following infection with M. tuberculosis was impaired in mice that had been exposed to M. avium. Our data suggest that exposure to environmental mycobacteria can negatively impact the protection afforded by BCG. These findings are relevant for the development of a vaccine administered in regions with elevated levels of environmental mycobacteria.     

Multiplex PCR-identified cutaneous tuberculosis evoked by Mycobacterium bovis BCG vaccination in a healthy baby

This is the first identified case of Mycobacterium bovis bacillus Calmette-Guerin (BCG)-derived cutaneous tuberculosis that localizes at a place different from the vaccination site in hosts without immune deficiency. A healthy baby with a developing abscess is described. A multiplex PCR identified the abscess as originating from M. bovis BCG Tokyo 172.     

Immunological memory in CBA and CBA/N mice after vaccination with Mycobacterium bovis (BCG)

In inbred CBA and CBA/N mice immunological memory was induced by subcutaneous injection of Mycobacterium bovis (BCG). Experiments with adoptive transfer of spleen T cells and ionomycin-resistant T cells (memory cells) between CBA and CBA/N mice in various combinations showed that immunological memory was not formed in CBA/N mice, but can be induced by adoptive transfer of cells from CBA mice.     

A novel tumor necrosis factor (TNF) mimetic peptide prevents recrudescence of Mycobacterium bovis bacillus Calmette-Guerin (BCG) infection in CD4+ T cell-depleted mice

Tumor necrosis factor (TNF) is required to control mycobacterial infections, but its therapeutic value is limited by its in vivo instability and toxicity. The efficacy of a nontoxic TNF-mimetic peptide (TNF70-80) was tested in mice infected with Mycobacterium bovis bacillus Callette-Guerin (BCG). In vitro TNF70-80 and recombinant human TNF (hTNF) acted with interferon gamma (IFN-gamma) to reduce bacterial replication and to induce synthesis of bactericidal nitric oxide (NO) in BCG-infected, bone marrow-derived murine macrophages. The dose-dependent inhibitory effect on bacterial replication was blocked by neutralizing anti-IFN-gamma and anti-hTNF mAbs. Further, n-monomethyl-L-arginine (n-MMA) and a soluble TNF-receptor I (TNFRI-IgG) blocked bacterial growth and NO synthesis. Therefore, the peptide acted with IFN-gamma via induction of NO synthase and signaled through TNFRI receptors. Concomitant in vivo treatment with TNF70-80 or hTNF prevented reactivation of chronic BCG infection in mice depleted of CD4+ T cells by injecting anti-CD4 antibodies. Granuloma number and bacterial load were comparable in treated, T cell-depleted mice and in chronically infected, intact animals. Thus, TNF70-80 and hTNF can modulate recrudescent BCG infection in CD4+ T cell-deficient mice.     

Activated human epitope-specific T cells identified by class II tetramers reside within a CD4high, proliferating subset

Antigen-specific T cells acquire a distinctive phenotype during activation, with characteristic acquisition of surface markers and patterns of gene expression. Early after antigen stimulation, CD4(+) T lymphocytes increase their surface density of the CD4 marker, a trait which has been used to identify antigen-activated cells. The recent development of MHC tetramer technologies has greatly improved the ability to detect HLA class I-restricted T cells specific for known antigen epitopes. We have recently extended these studies to human class II-restricted CD4(+) T cell responses and now describe antigen-specific T cell responses from human peripheral blood in which elevated CD4 expression levels in human T cells following antigen stimulation identify the activated and proliferating subset of cells. The CD4(high) population is substantially enriched in epitope-specific cells identified by class II tetramer staining and almost all tetramer-positive cells are contained within the CD4(high) population. T cell clones derived from the tetramer-positive, CD4(high) population demonstrate antigen specificity and maintain tetramer staining, while the substantial number of CD4(high) cells which fail to stain with tetramer appear to proliferate as a result of bystander activation. Epitope-specific components of a polyclonal immune response are directly visualized and quantitated by tetramer detection, providing a direct measure of the heterogeneity of the human immune response.     

Abnormalities within CD4 and CD8 T lymphocytes subsets in type 1 (insulin-dependent) diabetes.

Abnormalities in the proportions of various T lymphocyte subpopulations have been found in a number of autoimmune diseases. Monoclonal antibodies labelled with various fluorochromes were used here to define the percentages of subsets, and especially to divide CD4+ (helper/inducer) and CD8+ (suppressor/cytotoxic) cells into phenotypic subgroups. Blood samples were analysed from 25 patients (age 10.1 +/- 3.7 years) with recently diagnosed insulin-dependent diabetes mellitus (IDDM) and 25 age- and sex-matched control subjects. The percentages of CD4+ cells and CD4+CD45RA+ cells described as naive T helper cells or suppressor/inducers were increased in the IDDM patients (P less than 0.05 and P less than 0.05. Student's t-test, respectively), whereas the percentage of CD4+CD45RA- cells (memory T-helper cells, helper/inducers) was similar in the patients and controls. The percentage of CD8+CD11b+ cells containing suppressor/effector lymphocytes was decreased in the IDDM patients as compared with the controls (P less than 0.01) but no significant difference was seen in total CD8+ cells. The percentages of CD3+ cells and the proportions of these simultaneously positive for HLA-DR antigen (activated T cells) were also increased in the recent IDDM patients (P less than 0.001 and P less than 0.05, respectively), while the proportion of CD20+ B cells was decreased (P less than 0.05). The findings support the view that disturbed immune regulation occurs in IDDM and indicate that further division of T cell subpopulations may clarify our understanding of the disease process.     

Prophylactic effect of mycobacterium bovis BCG vaccination against osteomyelitis in children with Mycobacterium ulcerans disease (Buruli Ulcer)

Mycobacterium ulcerans disease, or Buruli ulcer (BU), causes significant morbidity in West Africa. In 233 consecutive, laboratory-confirmed samples from BU patients in Benin whose Mycobacterium bovis BCG scar status was known, 130 children (<15 years old) and 75 adults had a neonatal BCG vaccination scar. Of 130 children with BCG scars, 10 (7.7%) had osteomyelitis, while 3 of 9 children without BCG scars (33.3%) had osteomyelitis. Our observations support the conclusion that having a BCG vaccination scar provides significant protection against M. ulcerans osteomyelitis in children with BU disease.     

Requirements for CD4(+) T cell levels in acute Mycobacterium bovis strain bacille Calmette Guerin (BCG)-induced granulomas differ for optimal mycobacterial control versus granuloma formation

Bacille Calmette Guérin (BCG)-induced granulomas contain T cells that express a broad TCR repertoire even at the level of the individual lesion. We have developed a BCG infection model in mice having only one T cell specific for a recombinant BCG epitope expressed in a lipoprotein fusion protein. Here we report that the single T cell model induces well-formed granulomas, but has weaker protection than that conferred by wild-type granulomas. This finding correlates with lower CD4(+) T cell recruitment into acute granulomas (3 weeks post infection). Chronic granulomas (6 weeks post infection) contain similar proportions of CD4(+) T cells in both models, but in the single T cell model the proportion of leukocyte function-associated antigen-1 low, non-IFNgamma-producing CD4(+) T cells is lower. In fact, even though it is likely that there are very few, if any, IFNgamma(+) CD4(+) T cells present in the single T cell model, granuloma integrity is not influenced, indicating that high levels of IFNgamma are not required for granuloma maintenance. These data underline the importance of early CD4(+) T cell recruitment into the granuloma to anti-mycobacterial protection and show that CD4(+) T cell levels required for granuloma formation and optimal protection are different. These data also show that T cell repertoire complexity contributes to protection against mycobacteria.     

Culture filtrate specific H-2(b) restricted CD8+ T cells activated in vivo by Mycobacterium tuberculosis or bovis BCG recognize a restricted number of immunodominant peptides

We previously demonstrated that Bacillus Calmette-Guerin (BCG) immunization activated D(b) restricted CD8+ cytolytic T lymphocyte (CTL) recognizing target cells incubated with mycobacterial culture filtrate. Here, we show that in vitro restimulation of spleen cells from BCG vaccinated or Mycobacterium tuberculosis infected mice with culture filtrate antigens leads to the appearance of a high percentage of D(b) restricted IFNgamma synthesizing CD8+ T cell blasts. Transporter associated protein-2 mutated RMA-S cells incubated with soluble culture filtrate proteins had their MHC class I D(b) but not K(b) molecules stabilized at the surface indicating that only D(b) ligands might be generated by antigen presenting cells. MHC class I bound peptides were acid eluted from the surface of RMA-S cells incubated with M. tuberculosis culture filtrate proteins. The crude peptide preparation was able to sensitize RMA-S cells for recognition by culture filtrate-specific cytolytic T cells. Peptides were subsequently fractionnated by reverse-phase high performance liquid chromatography and the main biological activity was identified in two fractions. These results provide a further evidence that the processing of exogenous culture filtrate proteins in vitro leads to the presentation of a restricted number or even a single immunodominant peptide to culture filtrate-specific CD8+ T cells.     

Immune responses induced in cattle by vaccination with a recombinant adenovirus expressing Mycobacterial antigen 85A and Mycobacterium bovis BCG

Cattle were vaccinated with an adenovirus expressing the mycobacterial antigen 85A (rAd85A), with Mycobacterium bovis BCG followed by rAd85A heterologous boosting, or with rAd85A followed by BCG boosting. BCG/rAd85A resulted in the highest direct gamma interferon responses. Cultured enzyme-linked immunospot assay analysis demonstrated that memory responses were induced by all three protocols but were strongest after BCG/rAd85A and rAd85A/BCG vaccination.     

Revaccination of neonatal calves with Mycobacterium bovis BCG reduces the level of protection against bovine tuberculosis induced by a single vaccination

Cattle may provide a suitable model for testing ways of improving tuberculosis vaccine efficacy in human infants. A vaccination and challenge study was undertaken in calves to determine the optimal time to vaccinate neonatal animals with Mycobacterium bovis bacillus Calmette-Guérin (BCG) for protection against tuberculosis and to determine whether revaccination with BCG was beneficial. Calves (10 per group) were vaccinated with BCG within 8 h of birth or at 6 weeks of age, when immune responses to antigens of environmental mycobacteria were detectable, or vaccinated at birth and revaccinated at 6 weeks. A control group was not vaccinated. BCG vaccination at birth induced strong antigen-specific gamma interferon (IFN-gamma) and interleukin-2 (IL-2) responses and antigen-specific activation in CD4(+), CD8(+), and WC1(+) gammadelta T-cell subsets from blood. The proportions of animals per group with macroscopic tuberculous lesions after challenge were 0/10 for BCG at birth, 1/9 for BCG at 6 weeks, 4/10 for the revaccinated group, and 10/10 for the nonvaccinated group. There was no significant difference in the levels of protection between groups vaccinated at birth or at 6 weeks, while animals vaccinated both at birth and at 6 weeks had significantly less protection than those vaccinated only at birth. The revaccinated calves that subsequently developed tuberculous lesions had significantly stronger IFN-gamma and IL-2 responses to bovine purified protein derivative after the BCG booster than those in the same group that did not develop lesions. The results indicated that BCG vaccination at birth induced a high level of immunity and that the sensitization of very young animals to antigens of environmental mycobacteria by 6 weeks of age did not affect the effectiveness of BCG. However, BCG revaccination of these young animals was contraindicated.     

HIV Tat蛋白增加CD4+ T淋巴细胞bcl-2表达并抑制TRAIL诱导的细胞凋亡

目的探讨HIV Tat蛋白对CD4^＋ T淋巴细胞bcl-2表达的影响，并探讨肿瘤坏死因子相关凋亡诱导配体（TRAIL）与经HIV Tat蛋白刺激后的CD4^＋ T淋巴细胞凋亡的相互关系。方法用Western blot方法测定经HIV Tat蛋白刺激后，CD4＋ T淋巴细胞表达bcl-2的水平，以λ-氨基放线菌素D（7-AAD）和AnnexinV染色方法测定CD4＋ T淋巴细胞的凋亡。结果HIV Tat蛋白能明显增加CD4＋ T淋巴细胞bcl-2的表达，以100ng/ml为最佳浓度；7-AAD染色结果表明100ng/ml重组TRAIL能诱导53．85％±2．63％的CD4^＋ T淋巴细胞发生凋亡，如CD4^＋ T淋巴细胞经HIV Tat蛋白刺激后，则仅有16．04％±5．26％的细胞发生凋亡，此作用能被多克隆抗-Tat蛋白抗体抑制。Annexin V染色取得了同样的结果。结论经HIV Tat蛋白刺激后CD4^＋ T淋巴细胞bcl-2的表达明显增加，并能抑制由重组TRAIL诱导的细胞凋亡，提示HIV Tat蛋白是导致HIV在CD4^＋ T淋巴细胞内持续感染的重要调节蛋白，在HIV感染中起十分重要作用。     

Protection by CD4 or CD8 T Cells against Pulmonary Mycobacterium bovis Bacillus Calmette-Guérin Infection

Mice deficient in CD8 T cells demonstrated levels of Th1 cytokines and granulomatous responses in the lungs very similar to those demonstrated by normal control mice and were fully capable of controlling pulmonary mycobacterial infection by Mycobacterium bovis BCG as assessed at day 37 postinfection. In comparison, mice deficient in CD4 T cells had similar levels of interleukin-12 (IL-12) and tumor necrosis factor alpha but lower levels of gamma interferon in the lungs and were still able to mount tissue granulomatous responses and control pulmonary mycobacterial infection. In contrast, IL-12^−/− mice with impaired CD4 and CD8 T-cell responses had a markedly weakened control of infection, whereas SCID mice deficient in all T cells succumbed to such pulmonary mycobacterial infections.     

Increased activity of FIM in serum of mice during a Mycobacterium bovis (BCG) infection.

In mice given an intravenous injection of Mycobacterium bovis (BCG), the bacilli proliferated in the spleen, liver and lungs but the peritoneal cavity remained sterile. The numbers of blood monocytes and alveolar macrophages were increased during the first 2 weeks of the infection, whereas the number of peritoneal macrophages remained constant. To study whether factor-increasing monocytopoiesis (FIM) plays a role in the regulation of the monocytosis during the BCG infection, the activity of this factor in the serum of mice at various intervals during the infection was determined. Previous studies have shown that FIM stimulates monocyte production by its effect on the mitotic activity of monoblasts and promonocytes in the bone marrow. The FIM activity of the serum reached a maximum on Day 4 and remained elevated during the first 21 days of the BCG infection. Since FIM is synthesized and secreted by macrophages that have phagocytosed opsonized particles, it is highly probable that FIM occurring in serum originates from macrophages that have ingested BCG. The results of the present study led to the conclusion that FIM plays a role in the monocytosis developing during infection with BCG.     

HIV/AIDS患者疾病进程与CD4+CD25hi调节性T细胞之间相关性研究

目的 通过分析不同阶段HIV感染者外周血CD4+CD25hi调节性T细胞(CD4+CD25hiregulatory T cells,Treg cells)与外周血免疫状态和病毒载量的相关性,探讨Treg细胞对HIV/AIDS发病进程的影响.方法 采集116例HIV感染者和21例正常人对照外周血,用4色流式细胞术进行CD4+和CD8+T细胞绝对数计数;用3色流式细胞术进行Treg细胞测定;用荧光定量PCR法进行HIVRNA载晕测定.实验数据用回归统计学方法和T检验方法进行分析.结果 HIV感染者外周血Treg细胞频率在HIV感染初期显著下降,之后随着疾病的进程逐渐升高.在CD4+T细胞大于300/μl的患者低于正常对照组,在CD4+T细胞小于100/μl的患者高于正常对照组,差异具有统计学意义.Treg细胞频率与CD4+T淋巴细胞绝对数和CD4+/CD8+之间均呈负相关.其相关系数r和P值各为r=-0.564,P＜0.001和r=-0.377,P＜0.001;Treg细胞频率与血浆HIV病毒载量呈正相关,其相关系数r=0.514.P＜0.001.结论 CD4+CD25hi Treg细胞可能是参与艾滋病免疫发病机理的重要细胞,在HIV感染发病进程的不同阶段具有不同的意义,其确切机制有待进行进一步研究.     

CD40 ligand (CD154) does not contribute to lymphocyte-mediated inhibition of virulent Mycobacterium tuberculosis within human monocytes

Human monocytes displayed increased expression of CD40 following infection with virulent Mycobacterium tuberculosis. Nevertheless, soluble CD40 ligand (CD40L; also designated CD154) had no effect on the intracellular growth of the organism. Restriction of the intracellular growth of M. tuberculosis by peripheral blood lymphocytes and antigen-specific CD4+ T-cell lines likewise was not reduced by blocking anti-CD40L monoclonal antibody 5c8.     

Continuous expression of I-A antigen by peritoneal macrophages from mice resistant to Mycobacterium bovis (strain BCG)

Monoclonal antibodies were used to monitor the expression of I-A antigen on the surface of macrophages obtained from mice immunized to Mycobacterium bovis (strain BCG). Unlike the transient nature of I-A expression by macrophages from Listeria-injected mice, peritoneal macrophages from mice injected 28 days previously with 10(4) BCG expressed I-A continuously. The continued expression was not due to the presence of antigen or of contaminating lymphocytes. When we compared the kinetics of I-A expression from different strains of mice, the continuous expression of I-A correlated with the genetic resistance of the mice to BCG. Macrophages from mice that were resistant to BCG expressed I-A continuously, while macrophages from BCG susceptible mice expressed I-A transiently. Injection of resistant mice with Salmonella typhimurium did not result in the induction of a population of macrophages that expressed I-A continuously. This suggests that the Bcg gene may not be the same as that responsible for resistance to Salmonella (Ity) or Leishmania (Lsh).