Elsevier

Vaccine

Volume 25, Issue 50, 5 December 2007, Pages 8384-8394
Vaccine

Mycobacterium bovis BCG vaccination induces memory CD4+ T cells characterized by effector biomarker expression and anti-mycobacterial activity

https://doi.org/10.1016/j.vaccine.2007.10.011Get rights and content

Abstract

The effector mechanisms used by CD4+ T cells to control mycobacteria differ between humans and rodent models of TB and should be investigated in additional animal models. In these studies, the bovine model was used to characterize the mycobactericidal CD4+ T cell response induced by vaccination with the attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG). Antigenic stimulation of peripheral blood CD4+ T cells from BCG-vaccinated cattle enhanced expression of perforin and IFNγ in cells expressing a CD45RACD45RO+CD62L+ cell surface phenotype, enhanced transcription of granulysin, IFNγ, perforin, IL-4, IL-13, and IL-21, and enhanced anti-mycobacterial activity of CD4+ T cells against BCG-infected macrophages.

Introduction

Tuberculosis (TB) is a global health emergency, affecting approximately one-third of the world's population [1], [2]. Development of vaccines and therapeutics to curtail its spread requires improved understanding of the protective immune response to TB [3], [4]. Immunity to TB is complex and includes components of both the innate and acquired immune systems. Cell mediated immune (CMI) responses are the most important correlate to protective immunity to TB in all animal models. The attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG), currently in global use as a human TB vaccine and used experimentally in cattle, elicits strong CMI responses despite variable efficacy in vaccinated populations [4], [5]. The widely accepted biomarkers of CMI responsiveness (IFNγ and lymphocyte proliferation) frequently fail to predict protective immunity to TB in adult populations [3]. Biomarkers of vaccine-induced protection against TB in small animal models are frequently not predictive of human immunity to TB [6], [7], [8]. Thus, there are currently no consistent immune correlates of vaccine-induced protection against TB [6], [7], [8]. These failures highlight the need to further characterize the cellular subsets and mechanisms of the CMI response, induced by BCG, that contribute to protective immunity to TB.

A substantial role for CD4+ T cells in protective immunity to TB is highlighted by the susceptibility of CD4-gene deleted mice and HIV-infected individuals [3]. A strong Type 1 cytokine bias by CD4+ T cells is thought to correlate with protective immunity against TB, with the degree of bias varying among model systems [9], [10]. Enhanced microbicidal activity of infected macrophages (Mϕ) through inducible nitric oxide synthase (iNOS) is predominantly mediated by IFNγ produced by CD4+ T cells and represents one of the most important protective mechanisms in the murine model of TB [3], [8]. In human TB, the protective role of CD4-derived IFNγ is not fully understood and may be independent of iNOS mediated killing of intracellular mycobacteria [3]. Alternative mechanisms of Mϕ elimination of intracellular M. tuberculosis (Mtb) include activation through the P2X7 purinergic receptor [11], [12], acquisition of apoptotic neutrophil granules [13], and induction of cathelicidin by 1, 25-dihydroxy vitamin D3 [14]. CD4+ T cells are the primary source of IL-21, a pleiotropic cytokine that enhances lytic activity of cytotoxic T cells and NK cells [15].

CTL activity by CD4+, as well as CD8+ and γδ T cells, may play an important role in human immunity to TB [16], [17], [18], [19]. Results from in vitro experiments using Mtb specific T cell lines and peripheral blood mononuclear cells from Mtb-infected PPD+ individuals demonstrate that cytotoxic activity of human CD4+ T cells may also play an important role in immunity to TB [20], [21]. Restriction of Mtb growth by human CD4+ T cells is granule dependent and independent of Fas/FasL interactions [20]. Granulysin lyses intracellular Mtb in a perforin dependent manner, and correlates to inhibitory activity of human T cells isolated from peripheral blood of subjects reactive to PPD [18], [22]. These preliminary studies in PPD+ individuals and human cells lines suggest that granulysin and perforin may have important utility as biomarkers of TB immunity induced by vaccination or exposure to Mtb. The murine cell mediated immune response to mycobacteria appears to be less reliant on cytotoxic granule proteins in general, as perforin and granzyme B deficient mice are not more susceptible to infection with Mtb compared to wild type mice [23]. In cattle, bactericidal responses in peripheral blood T lymphocytes have been described [24], [25], [26]. Based on the incorporation of 3H-Uracil by mycobacteria, these reports describe the mycobactericidal/mycobacteriostatic ability of total peripheral blood cells [24], CD4+[25] and CD8+ T cells [25], [26]. Importantly, it was shown the mycobactericidal/mycobacteriostatic activity of peripheral blood lymphocytes was independent of IFNγ [24].

Characterization of post-vaccination biomarkers in an animal model with clinical presentation similar to human TB, and similar expression of lytic granule molecules by T lymphocytes, represents an important avenue for investigation of protective immune responses to TB. Studies of the full complement of cytotoxic biomarkers in murine models are hindered by the lack of a granulysin gene homologue and a disproportionate reliance on IFNγ mediated Mϕ activation for containment of intracellular bacteria [3]. Cattle represent an important animal model of TB, with similar clinical presentation of disease and protective immune responses [5], [27], [28]. Studies of immunity to M. bovis infection in cattle and Mtb in humans demonstrate important similarities, including the relative importance and sources of IFNγ, the expression of memory markers by antigen specific T cells, the balance of Th1 and Th2 cytokines, and bacterial containment through T cell cytotoxicity [24], [25], [26], [29], [30], [31], [32], [33]. The level of pathology in bovine TB (bTB) correlates to the amount of IFNγ produced by peripheral blood cells cultured in vitro with mycobacterial antigens [34]. Bovine homologues of granulysin and perforin are inducible upon T cell activation and are expressed in the granuloma cells of M. bovis-infected cattle [35]. Very recently, the bovine model of TB has been utilized to screen candidate neonatal TB vaccines prior to non-human primate studies [36].

The objective of the current work was to evaluate antigen specific recall of cytokine and lytic protein biomarkers in purified bovine CD4+ T cells from peripheral blood following vaccination with BCG. Our results demonstrate that vaccination of cattle with BCG induces a subpopulation of mycobacteria specific CD4+ T cells that are characterized by the expression of a cell-surface memory phenotype, enhanced expression of cytokines and mycobactericidal molecules, and anti-mycobacterial activity against intracellular BCG. These results, and similar studies in our laboratories characterizing CD8+ T cell effector molecules, will allow for significant advancement in the use of the bovine model of TB to correlate in vitro immune biomarkers to protection from challenge in experimentally controlled vaccination programs.

Section snippets

Animals

All animal experiments were carried out following local and National guidelines on the use of animals for experimentation and with the approval of local and National Ethics Committees. Peripheral blood samples used in these studies were obtained from BCG-vaccinated and non-vaccinated cattle housed at the Institute for Animal Health (Compton, UK) and the National Animal Disease Center (Ames, IA). All animals were vaccinated s.c. with 106 CFU of BCG. Animals were vaccinated with a second dose of 10

Functional characterization of effector and memory CD4+ T cells following vaccination

A recall CMI response to mycobacterial antigens was measurable in a whole blood IFNγ assay (Fig. 1A) 6 weeks after vaccination with M. bovis BCG (BCG). It has been shown that CD4+ T cells are the main cell population secreting IFNγ in PBMC from mycobacteria-sensitized cattle following stimulation in vitro[39]. Accordingly, we isolated CD4+ T cells and sought to determine whether the response elicited following antigen presentation was influenced by the physical form of the antigen. Fig. 1B

Discussion

Major roadblocks to the design of vaccines that protect against TB are lack of surrogate markers of protection and lack of suitable animal models [4], [40]. Understanding the mechanisms involved in the development of protective CD4+ T cell memory responses is an important component of TB vaccine enhancement efforts. Thus, there is currently a need to characterize biomarkers of CD4+ T cell mediated immunity in additional animal models of TB. In the present study, we used the bovine model of TB

Acknowledgments

The authors thank the personnel of the animal facilities at IAH Compton and the National Animal Disease Center. We also thank Dan Pong for technical assistance in real-time PCR assays. We thank the TX/UK Collaborative Research Initiative for sponsoring the travel grants to initiate this effort among three laboratories in two countries. This work was supported by the NIAID Fellowship for Training in Emerging and Re-emerging Infectious Disease NIAID T32AI07536 and the Sealy Center for Vaccine

References (58)

  • J.L. Flynn

    Lessons from experimental Mycobacterium tuberculosis infections

    Microbes Infect

    (2006)
  • D.M. Estes et al.

    Type 1 and type 2 responses in regulation of Ig isotype expression in cattle

    Vet Immunol Immunopathol

    (2002)
  • M.R. Foote et al.

    Effects of age and nutrition on expression of CD25, CD44, and L-selectin (CD62L) on T-cells from neonatal calves

    J Dairy Sci

    (2005)
  • W.C. Brown et al.

    Bovine type 1 and type 2 responses

    Vet Immunol Immunopathol

    (1998)
  • A.S. Fauci et al.

    Emerging infectious diseases: a 10-year perspective from the National Institute of Allergy and Infectious Diseases

    Emerg Infect Dis

    (2005)
  • P. Nunn et al.

    Tuberculosis control in the era of HIV

    Nat Rev Immunol

    (2005)
  • S.H. Kaufmann

    Envisioning future strategies for vaccination against tuberculosis

    Nat Rev Immunol

    (2006)
  • B.M. Buddle et al.

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

    Infect Immun

    (2003)
  • D.F. Hoft et al.

    Investigation of the relationships between immune-mediated inhibition of mycobacterial growth and other potential surrogate markers of protective Mycobacterium tuberculosis immunity

    J Infect Dis

    (2002)
  • S.H. Kaufmann

    How can immunology contribute to the control of tuberculosis?

    Nat Rev Immunol

    (2001)
  • J.L. Flynn et al.

    Immunology of tuberculosis

    Annu Rev Immunol

    (2001)
  • I.P. Fairbairn et al.

    ATP-mediated killing of intracellular mycobacteria by macrophages is a P2X(7)-dependent process inducing bacterial death by phagosome-lysosome fusion

    J Immunol

    (2001)
  • B.H. Tan et al.

    Macrophages acquire neutrophil granules for antimicrobial activity against intracellular pathogens

    J Immunol

    (2006)
  • P.T. Liu et al.

    Cutting edge: vitamin D-mediated human antimicrobial activity against Mycobacterium tuberculosis is dependent on the induction of cathelicidin

    J Immunol

    (2007)
  • W.J. Leonard et al.

    Interleukin-21: a modulator of lymphoid proliferation, apoptosis and differentiation

    Nat Rev Immunol

    (2005)
  • S. Cho et al.

    Antimicrobial activity of MHC class I-restricted CD8+ T cells in human tuberculosis

    Proc Natl Acad Sci USA

    (2000)
  • F. Dieli et al.

    Granulysin-dependent killing of intracellular and extracellular Mycobacterium tuberculosis by Vgamma9/Vdelta2 T lymphocytes

    J Infect Dis

    (2001)
  • S. Stenger et al.

    An antimicrobial activity of cytolytic T cells mediated by granulysin

    Science

    (1998)
  • S. Stenger et al.

    Differential effects of cytolytic T cell subsets on intracellular infection

    Science

    (1997)
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