Mycobacterium bovis BCG Vaccination Induces Divergent Proinflammatory or Regulatory T Cell Responses in Adults

Mycobacterium bovis bacillus Calmette-Guérin (BCG), the only currently available vaccine against tuberculosis, induces variable protection in adults. Immune correlates of protection are lacking, and analyses on cytokine-producing T cell subsets in protected versus unprotected cohorts have yielded inconsistent results. We studied the primary T cell response, both proinflammatory and regulatory T cell responses, induced by BCG vaccination in adults. Twelve healthy adult volunteers who were tuberculin skin test (TST) negative, QuantiFERON test (QFT) negative, and BCG naive were vaccinated with BCG and followed up prospectively. BCG vaccination induced an unexpectedly dichotomous immune response in this small, BCG-naive, young-adult cohort: BCG vaccination induced either gamma interferon-positive (IFN-γ⁺) interleukin 2-positive (IL-2⁺) tumor necrosis factor α-positive (TNF-α⁺) polyfunctional CD4⁺ T cells concurrent with CD4⁺ IL-17A⁺ and CD8⁺ IFN-γ⁺ T cells or, in contrast, virtually absent cytokine responses with induction of CD8⁺ regulatory T cells. Significant induction of polyfunctional CD4⁺ IFN-γ⁺ IL-2⁺ TNF-α⁺ T cells and IFN-γ production by peripheral blood mononuclear cells (PBMCs) was confined to individuals with strong immunization-induced local skin inflammation and increased serum C-reactive protein (CRP). Conversely, in individuals with mild inflammation, regulatory-like CD8⁺ T cells were uniquely induced. Thus, BCG vaccination either induced a broad proinflammatory T cell response with local inflammatory reactogenicity or, in contrast, a predominant CD8⁺ regulatory T cell response with mild local inflammation, poor cytokine induction, and absent polyfunctional CD4⁺ T cells. Further detailed fine mapping of the heterogeneous host response to BCG vaccination using classical and nonclassical immune markers will enhance our understanding of the mechanisms and determinants that underlie the induction of apparently opposite immune responses and how these impact the ability of BCG to induce protective immunity to TB.     

Stimulation of human peripheral blood mononuclear cells with live Mycobacterium bovis BCG activates cytolytic CD8+ T cells in vitro

Experimental data have shown that Mycobacterium tuberculosis can survive within the host cell and in doing so may release secreted antigen into the endogenous antigen-processing pathway. If mycobacterial antigen can gain access to MHC class I molecules then CD8⁺ T cells may play a role in host defence against M. tuberculosis infection. To identify whether there is a role for the CD8⁺ T cell in mycobacterial infection we have stimulated peripheral blood mononuclear cells (PBMC) from bacillus Calmette–Guérin (BCG) vaccinated individuals with live M. bovis BCG. The activation state of the T cells was established by staining for the interleukin-2 (IL-2) receptor (CD25), HLA-DR or the transferrin receptor (CD71). Using FACScan analysis we have shown that, in vitro, live M. bovis BCG activates significantly more CD8⁺ T cells in comparison to the soluble antigen purified protein derivative (PPD). In addition, live M. bovis BCG activates more CD8⁺ T cells than a non-viable preparation of the same M. bovis BCG following irradiation. The function of the activated CD8⁺ T cells was addressed using positively selected cells in a cytotoxic T-cell assay. CD8⁺ T cells isolated from a 7-day M. bovis BCG-stimulated PBMC culture were shown to be cytolytic against target cells infected with live M. bovis BCG, dead M. bovis BCG, and to a lesser extent, PPD. These results suggest that CD8⁺ T cells may be activated by stimulation with live mycobacteria, and that this subset can play a cytolytic role in the immune response to mycobacterial infections.     

Exposure to human alveolar lining fluid enhances Mycobacterium bovis BCG vaccine efficacy against Mycobacterium tuberculosis infection in a CD8+ T-cell-dependent manner

Current tuberculosis (TB) treatments include chemotherapy and preventative vaccination with Mycobacterium bovis Bacillus Calmette-Guérin (BCG). In humans, however, BCG vaccination fails to fully protect against pulmonary TB. Few studies have considered the impact of the human lung mucosa (alveolar lining fluid (ALF)), which modifies the Mycobacterium tuberculosis (M.tb) cell wall, revealing alternate antigenic epitopes on the bacterium surface that alter its pathogenicity. We hypothesized that ALF-induced modification of BCG would induce better protection against aerosol infection with M.tb. Here we vaccinated mice with ALF-exposed BCG, mimicking the mycobacterial cell surface properties that would be present in the lung during M.tb infection. ALF-exposed BCG-vaccinated mice were more effective at reducing M.tb bacterial burden in the lung and spleen, and had reduced lung inflammation at late stages of M.tb infection. Improved BCG efficacy was associated with increased numbers of memory CD8⁺ T cells, and CD8⁺ T cells with the potential to produce interferon-γ in the lung in response to M.tb challenge. Depletion studies confirmed an essential role for CD8⁺ T cells in controlling M.tb bacterial burden. We conclude that ALF modifications to the M.tb cell wall in vivo are relevant in the context of vaccine design.     

Loss of Neurological Disease HSAN-I-Associated Gene SPTLC2 Impairs CD8+ T Cell Responses to Infection by Inhibiting T Cell Metabolic Fitness

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Specific Decrease in B-Cell-Derived Extracellular Vesicles Enhances Post-Chemotherapeutic CD8+ T Cell Responses

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CD137 is a Useful Marker for Identifying CD4+ T Cell Responses to Mycobacterium tuberculosis

Upregulation of CD137 on recently activated CD8⁺ T cells has been used to identify rare viral and tumour antigen‐specific T cells from the peripheral blood. We aimed to evaluate the accuracy of CD137 for identifying Mycobacterium tuberculosis (Mtb)‐reactive CD4⁺ T cells in the peripheral blood of infected individuals by flow cytometry and to investigate the characteristics of these CD137⁺CD4⁺ T cells. We initially enrolled 31 active tuberculosis (TB) patients, 31 individuals with latent TB infection (LTBI) and 25 healthy donors. The intracellular CD137 and interferon‐γ (IFN‐γ) production by CD4⁺ T cells was simultaneously detected under unstimulated and CFP10‐stimulated (culture filtrate protein 10, a Mtb‐specific antigen) conditions. In unstimulated CD4⁺ T cells, we found that the CD137 expression in the TB group was significantly higher than that in the LTBI group. Stimulation with CFP10 largely increased the CD4⁺ T cell CD137 expression in both the TB and LTBI groups. After CFP10 stimulation, the frequency of CD137⁺CD4⁺ T cells was higher than that of IFN‐γ⁺CD4⁺ T cells in both the TB and LTBI groups. Most of the CFP10‐activated IFN‐γ‐secreting cells were CD137‐positive, but only a small fraction of the CD137‐positive cells expressed IFN‐γ. An additional 20 patients with TB were enrolled to characterize the CD45RO⁺CCR7⁺, CD45RO⁺CCR7⁻ and CD45RO⁻ subsets in the CD137⁺CD4⁺ T cell populations. The Mtb‐specific CD137⁺CD4⁺ T cells were mainly identified as having an effector memory phenotype. In conclusion, CD137 is a useful marker that can be used for identifying Mtb‐reactive CD4⁺ T cells by flow cytometry.     

CD8+ T Cell Subsets in Aging

Infections are a major cause of illness and death amongst elderly people. Peripheral blood CD8⁺ T lymphocytes -which play a crucial role in host defence against viral infections-, are divided in subsets based upon the expression of several cell and activation markers. Since in senescence changes in peripheral blood CD8⁺ T lymphocyte compartment have been described, studies were performed to determine whether in aging there are variations in the peripheral blood CD8⁺CD38⁺, CD8⁺CD57⁺, CD8⁺HLA-DR⁺, CD8⁺CD45RA⁺ and CD8⁺CD45RO⁺ cell subset. A decrease in the CD8⁺CD45RA⁺ lymphocytes was observed, indicating that variations in the CD8⁺ compartment can take place with ageing.     

CD8+ T Cell Subsets in Aging

Infections are a major cause of illness and death amongst elderly people. Peripheral blood CD8⁺ T lymphocytes -which play a crucial role in host defence against viral infections-, are divided in subsets based upon the expression of several cell and activation markers. Since in senescence changes in peripheral blood CD8⁺ T lymphocyte compartment have been described, studies were performed to determine whether in aging there are variations in the peripheral blood CD8⁺CD38⁺, CD8⁺CD57⁺, CD8⁺HLA-DR⁺, CD8⁺CD45RA⁺ and CD8⁺CD45RO⁺ cell subset. A decrease in the CD8⁺CD45RA⁺ lymphocytes was observed, indicating that variations in the CD8⁺ compartment can take place with ageing.     

Monofunctional and Polyfunctional CD8+ T Cell Responses to Human Herpesvirus 8 Lytic and Latency Proteins

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi''s sarcoma, primary effusion lymphoma, and multicentric Castleman''s disease. It is postulated that CD8⁺ T cell responses play an important role in controlling HHV-8 infection and preventing development of disease. In this study, we investigated monofunctional and polyfunctional CD8⁺ T cell responses to HHV-8 lytic proteins gB (glycoprotein B) and K8.1 and latency proteins LANA-1 (latency-associated nuclear antigen-1) and K12. On the basis of our previous findings that dendritic cells (DC) reveal major histocompatibility complex (MHC) class I epitopes in gB, we used a DC-based system to identify 2 novel epitopes in gB, 2 in K8.1, 5 in LANA-1, and 1 in K12. These new HHV-8 epitopes activated monofunctional and polyfunctional CD8⁺ T cells that produced various combinations of gamma interferon, interleukin 2, tumor necrosis factor alpha, macrophage inhibitory protein 1β, and cytotoxic degranulation marker CD107a in healthy HHV-8-seropositive individuals. We were also able to detect HHV-8-specific CD8⁺ T cells in peripheral blood samples using HLA A*0201 pentamer complexes for one gB epitope, one K8.1 epitope, two LANA-1 epitopes, and one K12 epitope. These immunogenic regions of viral lytic and latency proteins could be important in T cell control of HHV-8 infection.Human herpesvirus 8 (HHV-8), also referred to as Kaposi''s sarcoma-associated herpesvirus, is a gammaherpesvirus that causes Kaposi''s sarcoma (KS), primary effusion lymphoma, and multicentric Castleman''s disease. The importance of developing effective prevention and treatment for HHV-8 infection is evident in that KS, a neoplasm of endothelial origin, continues to be the most common cancer among human immunodeficiency virus (HIV)-infected patients (8). KS is also the leading cause of cancer in children in sub-Saharan Africa (7). Although the incidence of KS in HIV-infected persons declined with the advent of antiretroviral therapy (ART) (10), KS can occur in persons on ART with suppressed HIV infection and high CD4⁺ T cell counts (25).The immune responses responsible for controlling HHV-8 infection and preventing KS are not clear. CD8⁺ T cell immunity likely plays a significant role in HHV-8 infection, as these cells have been shown to be crucial in controlling infection caused by the other human gammaherpesvirus, i.e., Epstein-Barr virus (EBV) (11, 14). In support of this hypothesis, our laboratory (40-42) and others (4-6, 12, 19, 23, 26-28, 31, 32, 36, 37, 43, 44) have shown that CD8⁺ T cells produce gamma interferon (IFN-γ) in response to HHV-8 immunodominant epitopes presented by major histocompatibility complex class I (MHC-I) in HHV-8-seropositive individuals. Little is known whether T cells produce other immune mediators in response to HHV-8 infection. Indeed, polyfunctional T cells, i.e., single cells producing two or more immune mediators, have been linked to control of HIV and other persistent infections (1, 24, 29, 33) and could play a role in controlling HHV-8 infection. In one recent study, HHV-8 epitope-specific, polyfunctional T cells were detected in patients with multicentric Castleman''s disease, but these cells did not differ in number from those in healthy controls (13). Another study has found that patients with controlled KS had HHV-8-specific CD8⁺ T cells that secreted IFN-γ and tumor necrosis factor alpha (TNF-α) but that patients with progressive disease had weaker and less polyfunctional CD8⁺ T cells (2).HHV-8 epitope-specific monofunctional and polyfunctional T cell immunity could be important in development of HHV-8 vaccines that induce T cell responses that target these viral epitopes. In the present study, we therefore investigated CD8⁺ T cell responses to two HHV-8 lytic proteins, gB (glycoprotein B) and K8.1, and two latency proteins, LANA-1 (latency associated nuclear antigen-1) and K12. We previously showed that optimal induction of T cell reactivity to the HHV-8 protein gB required 1 week of stimulation with peptide-loaded, autologous, mature, monocyte-derived dendritic cells (DC) (40). Using this enhanced DC-T cell stimulation system, we now have revealed several new epitopes for these four lytic and latency HHV-8 proteins in healthy HHV-8-seropositive individuals, which induce both monofunctional and polyfunctional CD8⁺ T cells. These regions of HHV-8 could be critical in understanding HHV-8 immunopathogenesis and in vaccine development.     

Activated CD4+ and CD8+ T Cell Proportions in Multiple Sclerosis Patients

The goal of this study was to trace the course of multiple sclerosis (MS) by evaluating the lymphocyte subpopulation counts and the levels of CD4+ and CD8+ T cell activation using flow cytometry. Samples obtained from healthy subjects (N?=?40) and patients with MS (N?=?290) were analyzed. Lymphocytes were labeled for the surface markers CD4+, CD8+, CD3+, CD16+, CD19+, CD45+, and CD53+ and the activation marker HLA-DR+. Cell counts were then determined using flow cytometry. A high degree of inter-individual variability was observed in the counts of all lymphocyte subtypes in the MS group. A significantly lower proportion of CD3+ T cells (69?±?14 % in healthy subjects and 60?±?17 % as a percent of total lymphocytes in MS patients), CD4+ T cells (41?±?11 and 28?±?18 %, respectively), and a significantly higher proportion of NK T cells (12?±?5 and 25?±?21 %, respectively) were observed in patients with MS than in healthy subjects. These differences led to a lowered CD4+/CD8+ T cell ratio. Furthermore, a significantly lower proportion of activated CD4+ T cells (HLA-DR+ CD4+; from 48?±?10 to 38?±?15 % as a percent of CD4+ cells) was observed in patients with MS than in healthy subjects. The high level of inter-individual variability in lymphocyte cell counts and the counts of activated T cells suggest that MS is a complex and heterogeneous disease.     

CD4+ CD8+ T Cell Reference Values in the Mexico City Population

Due to the importance of determining the proportions of lymphocyte subpopulations in Mexicans as reference values for flow cytometry, the aim of this study was to establish CD4⁺ and CD8⁺ T cell reference values for healthy Mexicans according to gender and age. Our results may serve as reference standards for the Mexican city population.     

Mycobacterium tuberculosis-specific CD8+ T cells and their role in immunity

There are more cases of tuberculosis in the world today than at any other time in history. The global epidemic has generated intense interest into the immunological mechanisms that control infection. Although CD4+ T cells play a critical role in host immunity to Mycobacterium tuberculosis, there is considerable interest in understanding the role of other T cell subsets in preventing disease development following infection. CD8+ T cells are required for optimum host defense following M. tuberculosis infection, which has led to investigation into how this protective effect is mediated. A critical review of recent literature regarding the role of CD8+ T cells in protective immunity to M. tuberculosis infection is now required to address the strengths and weaknesses of these studies. In this article, we evaluate the evidence that CD8+ T cells are critical in immunity to M. tuberculosis infection. We discuss the specific mycobacterial proteins that are recognized by CD8+ T cells elicited during infection. Finally, we examine the effector mechanisms of CD8+ T cells generated during infection and synthesize recent studies to consider the protective roles that these T cells serve in vivo.     

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.     

Fatal Mycobacterium bovis BCG infection in TNF-LT-alpha-deficient mice

Neutralization of TNF or disruption of TNF-R1 leads to fatal Mycobacterium bovis BCG infection. Here we used TNF-LT-alpha-deficient mice to test whether a complete disruption of TNF and LT-alpha reduces further host resistance to BCG infection. The bacterial burden especially in the lungs of TNF-LT-alpha-deficient mice was significantly increased and the mice succumbed to infection between 8 and 10 weeks. In the absence of TNF-LT-alpha the granulomatous response was severely impaired and delayed. The cells in the granulomas of TNF-LT-alpha-deficient mice expressed low levels of MHC class II and ICAM-1. They contained a few T cells and F4/80-positive macrophages expressing little iNOS and acid phosphatase activity. By contrast, the lethal action of endotoxin was dramatically reduced in BCG-infected TNF-LT-alpha-deficient mice. In summary, in the absence of TNF-LT-alpha the recruitment and activation of mononuclear cells in response to BCG infection were significantly delayed and reduced resulting in immature granulomas allowing uncontrolled fatal infection.     

Antibody Response in Rabbits to Mycobacterium bovis BCG

The specificity of the immune response after immunization with Mycobacterium bovis BCG was studied by crossed immunoelectrophoresis with intermediate gel in a BCG/anti-BCG system, in which the reaction against thirty distinct components of BCG was recorded. After a single injection of total sonicate of 3 mg (dry weight) bacilli, the antibody response was markedly similar in eight rabbits. Th earliest and strongest response was directed against nine components of BCG; all but one of these belonged to the group of thirteen components that cross-react extensively with other mycobacteria. After repeated immunization with sonicate from about 0.8 microgram of BCG bacilli, five components still induced a marked antibody response. All but one of these components are among the most widely cross-reacting BCG components, and the observations made after subsequent challenge with the higher dose of BCG indicate that low-zone tolerance was induced against other components of the bacilli. The implication of these findings concerning formation of anti-mycobacterial antibodies in normal individuals and during mycobacterial infection is discussed.