THE ROLE OF α4β1 INTEGRIN (VLA-4) IN RECRUITMENT OF
MYCOBACTERIUM TUBERCULOSIS-SPECIFIC TH1-LIKE RECALL
RESPONSES TO THE HUMAN LUNG
by
JESSICA R. WALRATH
Submitted in partial fulfillment of the requirements
For the degree of Master of Science
Thesis Advisor: Dr. Richard Silver
Department of Pathology
CASE WESTERN RESERVE UNIVERSITY
January, 2008
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis of
______
candidate for the Master of Science Degree*.
(signed)______(chair of the committee)
______
______
______
______
______
(date) ______
*We also certify that written approval has been obtained for any proprietary material contained therein. TABLE OF CONTENTS
TABLE OF CONTENTS…………………………………….………………………….ii
LIST OF FIGURES………………………………………………..……………………iii
LIST OF ABBREVIATIONS…………………………………..………………………iv
ABSTRACT………………………………………………………...…………………….v
INTRODUCTION……………………………………………………………………….1
Overview……………………………………………………………………………...1 Pathogenesis of infection with M. tuberculosis……………….……………………...2 Lymphocyte homing and localization of protective immune responses……………..4 Dendritic cells in the development of tissue-specific homing……………………...... 6 Limitations of BCG vaccination……………………………………………………...7 Assessment of local immunity in humans following respiratory exposure to M. tuberculosis……………………………………………………………………….8
MATERIALS AND METHODS………………………………………………………11
RESULTS……………………………………………………………………………….21
Bronchoscopic challenge with PPD results in development of localized lymphocytic inflammation in challenged BAL segments…………………………..21 Skewing of homing molecule expression on CD4+ T-cells in baseline BAL………22 Role of homing molecule expression in recruitment of antigen-specific Th1 cells to the lung in response to bronchoscopic challenge with PPD…………...24 Assessment of the role of lymphocyte proliferation in increasing BAL CD4+ T-cells in response to PPD challenge…………………………………….…..26
DISCUSSION………………………………………………………………………...…28
FIGURES…………………………………………………………………………..……36
REFERENCES……………………………………………………………………….…43
LIST OF FIGURES
Figure 1 Technique of bronchoscopic segmental antigen challenge………………..36
Figure 2 Lymphocyte subset recruitment in response to PPD
bronchoscopic challenge of PPD+ subjects…………………………….....37
Figure 3 Analysis of homing molecule expression on CD4+ T-cells in baseline
BAL, PBMC, and 48hrs post challenge BAL of a PPD+ subject………....38
Figure 4 A Percent expression of homing molecules on CD4+ T-cells
in baseline BAL and PBMC of PPD+ subjects……………………………39
Figure 4B Percent expression of homing molecules on CD4+ T-cells
in baseline BAL and PBMC of PPD- subjects.……………………………39
Figure 5A Percent expression of homing molecules on CD4+ T-cells
in baseline BAL and 48hrs post challenge BAL of PPD+ subjects……….40
Figure 5B Total number of CD4+ T-cell expression of homing molecules
in baseline BAL and 48hrs post challenge BAL of PPD+ subjects…...…..40
Figure 6A Percent expression of homing molecules on PPD-specific
Th1-like CD4+ T-cells in baseline BAL and 48hrs
post challenge BAL of PPD+ subjects…………………………………….41
Figure 6B Total number of PPD-specific Th1-like CD4+ T-cell expression of
homing molecules in baseline BAL and 48hrs post challenge
BAL of PPD+ subjects……...... 41
Figure 7 In vitro and in vivo Ki67 assay of baseline BAL, PBMC
and 48hrs post challenge BAL of PPD+ subjects……………………...….42
ii LIST OF ABBREVIATIONS
γδ gamma delta α4β1 alpha 4 beta 1 α4β7 alpha 4 beta 7 APC allophycocyanin AS autologous serum BAL bronchoalveolar lavage BCG Mycobacterium bovis strain Bacillus of Calmette and Guerin BrdU bromodeoxyuridine CCR chemokines (C-C motif) receptor CLA cutaneous lymphocyte antigen CXCR chemokines (C-X motif) receptor DC dendritic cell DTH delayed-type hypersensitivity EDTA ethylenediaminetetraacetic acid FITC fluorescein isothicoyanate HIV human immunodeficiency virus IFNγ interferon gamma IL interleukin IMDM Iscove’s Modified Dulbecco’s Medium IP-10 interferon gamma-inducible protein 10 MAdCAM-1 mucosal addressin cell adhesion molecule 1 Mig monokine induced by interferon gamma MIP-1α macrophage inflammatory protein-1-alpha NK natural killer NS normal saline PBMC peripheral blood mononuclear cells PBS phosphate buffer saline PE phycoerythrin PerCP peridinin chlorophyll protein PFA paraformaldyde PPD purified protein derivative of Mycobacterium tuberculosis RANTES regulated-upon activation, normal T-cell expressed and secreted RML right middle lobe SEB staphylococcal enterotoxin B Th1 T-helper (aka CD4+ T-cell), type 1, IFNγ and IL-2 producing Th2 T-helper (aka CD4+ T-cell), type 2, IL-4 producing TU tuberculin units VCAM-1 vascular cell adhesion molecule 1 VLA-4 very late antigen 4 (also known as α4β1)
iii The Role of α4β1 Integrin (VLA-4) in Recruitment of Mycobacterium tuberculosis-
Specific Th1-like Recall Responses to the Human Lung
Abstract
by
JESSICA R. WALRATH
Tuberculosis remains a major international health threat. Although BCG vaccination does not clearly protect against pulmonary tuberculosis, Mycobacterium tuberculosis- exposed individuals are relatively protected against subsequent re-infection. We have utilized bronchoscopic antigen challenge with PPD to model immune responses of infected individuals following re-exposure. Currently, we assessed the role of homing molecules in recruitment of M. tuberculosis-specific recall responses to the human lung.
Intracellular staining for IFNγ was performed, as was surface staining for α4β1 and α4β7 integrins and CLA. Baseline bronchoalveolar lavage (BAL) is enriched for α4β1- expressing CD4+ T-cells. This skewing continues following PPD-induced lymphocyte recruitment, and is even more pronounced for M. tuberculosis-specific CD4+ T-cells.
Expression of α4β1 integrin therefore appears to mediate optimal localization of recall responses to the lung. Comparison with vaccinated subjects may clarify whether intradermal BCG is as effective as natural infection in inducing recall responses that can be localized to the lung.
iv INTRODUCTION
Overview:
Mycobacterium tuberculosis currently infects over 1/3 of the world’s population. There are 8.4 million new cases of tuberculosis annually, and it is estimated that 1.5-2 million people die of tuberculosis each year [1]. Multiple factors contribute to the continued importance of tuberculosis as an international threat to public health. Although tuberculosis is largely a preventable and treatable disease, effective antibiotic therapy requires months of daily treatment. Delivering this therapy is difficult in underdeveloped countries where government infrastructure is often weak and tuberculosis control programs are poorly funded. Not surprisingly, these countries account for 95% of the tuberculosis cases and tuberculosis-related deaths worldwide [1]. The onset of the HIV epidemic has been associated with increased incidence of tuberculosis due both to the marked susceptibility of HIV-infected individuals to development of active disease, and to the prominence of HIV infection in the same underdeveloped regions where M. tuberculosis infection is endemic [2, 3]. In addition, the development of multi-drug resistant and extensively drug resistant isolates of M. tuberculosis raise further concern for the prospects of the return to an era in which tuberculosis was not a treatable disease
[1, 4].
All of these circumstances point to the potential importance of effective vaccination in the control of tuberculosis. The current tuberculosis vaccine, the Bacillus of Calmette
1 and Guerin (BCG), is an empirically attenuated strain of Mycobacterium bovis developed in the 1920’s and used extensively since that time. In most of the world, BCG is given to newborns via intradermal injection. BCG vaccination is known to be effective at preventing disseminated tuberculosis in very young children, but does not clearly prevent the development of pulmonary tuberculosis in adults [5]. Because pulmonary tuberculosis is the contagious form of the disease, however, it represents the manifestation of M. tuberculosis infection for which prevention is most needed in order to improve world-wide control of the disease [6].
Pathogenesis of infection with Mycobacterium tuberculosis:
The normal course of pulmonary infection with M. tuberculosis may provide insight into how to improve upon the protection induced by BCG. Mycobacterium tuberculosis is transmitted from patients with pulmonary tuberculosis via aerosolized “droplet nuclei” generated by coughing. Individuals who are in close proximity to a contagious patient then inhale these droplets. Once inhaled, the bacilli travel to the alveoli of the lungs whether they are phagocytosed by alveolar macrophages [7]. Because M. tuberculosis bacilli are uniquely well-suited to survival within alveolar macrophages, these cells cannot themselves control the intracellular pathogen. Instead, intracellular replication continues until infected macrophages die, releasing viable bacilli. Containment of infection then requires the development of an antigen-specific lymphocyte response to the organism. This is initiated by the action of specialized tissue dendritic cells (DC) that travel to the alveoli, phagocytose the bacilli at the site of infection and then carry the
2 organism back to the draining lymph nodes [8]. Mycobacterium tuberculosis bacilli are able to survive intracellularly in dendritic cells, in a similar fashion to alveolar macrophages by having evolved mechanisms that block immune responses. From the lymphatic system, the M. tuberculosis also enters the bloodstream, causing a phase of disseminated infection [6]. In individuals with defective cell-mediated immunity, infection can remain uncontrolled and result in primary tuberculosis, which often displays extra-pulmonary manifestations including lymphadenitis, disseminated disease (miliary tuberculosis) and meningitis [2, 6]. In immunologically intact individuals, however, infected dendritic cells stimulate naïve T-cells capable of recognizing M. tuberculosis antigens, resulting in the development of an antigen-specific immune response that eventually serves to contain the organism, typically two to six weeks after initial infection, resulting in a period of latent infection [7]. Cell-mediated immunity does not have the capacity to sterilize the body of all viable organisms, however. Accordingly, the body’s ability to contain the organism can subsequently break down, resulting in reactivation tuberculosis at any site to which the organism spread during dissemination.
The lung is the most common site of reactivation tuberculosis, and because it is the uniquely contagious form of the disease, the most important from the perspective of public health.
Antigen-specific cell-mediated immunity to M. tuberculosis is manifest clinically by the acquisition of skin-test responsiveness to purified protein derivative of M. tuberculosis
(PPD) and immunologically by the development of relative protection from subsequent respiratory exposure to M. tuberculosis [9]. The mechanisms by which cell-mediated
3 immunity contains initial infection with M. tuberculosis, and protects against subsequent re-infection are poorly understood, however. Although multiple lymphocyte populations have been shown to have the capacity to contribute to containment of M. tuberculosis
[10-14] both murine and human studies suggest a central role for IFNγ-producing Th1- like CD4+ T-cells in protective immunity to the organism. In animal studies, antigen- specific CD4+ T-cells are the essential component of effective passive transfer of protective immunity to M. tuberculosis [15], and early rapid mobilization of IFNγ producing CD4+ T-cells to the lung is correlated with the capacity to contain M. tuberculosis infection [16-19]. In humans, the importance of functioning Th1-like cells in protection is evidenced by the marked susceptibility of HIV-infected individuals to the development of active tuberculosis [20] and by the increased incidence of mycobacterial disease in individuals with defects in expression or function of IFNγ receptors [21, 22].
Lymphocyte homing and the localization of protective immune responses:
A protective immune response to an inhaled pathogen such as M. tuberculosis would ideally elicit a localized memory response that could contain the organism within the lung at the time of re-exposure [8]. Memory T-cells are able to provide such a response by preferentially circulating to specific tissue sites through a process described as
“homing”. In contrast to naïve lymphocytes that continuously re-circulate between blood and secondary lymphoid tissues until they encounter specific antigens presented by dendritic cells (DC), lymphocytes that have been presented their specific antigen by DC proliferate and differentiate into memory cells that acquire adhesion molecule homing
4 receptors [7, 8, 23]. Effector memory cells provide peripheral memory and are defined by their lack of CCR7 expression and a defined profile of cytokine responses [24]. These receptors direct the subsequent circulation of memory lymphocytes to specific tissue sites
[25].
The phenomenon of lymphocyte homing was first observed by Gowans’s team in rats and later in sheep by Cahill’s group of scientists. They found that when lymphocytes were taken from the local lymph nodes of one animal and transferred to the bloodstream of another, the lymphocytes did not circulate throughout the whole body but rather migrated back to the tissue from which they had originated [26, 27]. It has subsequently been determined that specific lymphocyte surface integrin molecules direct these cells to
“home” back from the bloodstream to a particular tissue in both animal and human studies [28-30].
The best characterized of these homing molecules are cutaneous lymphocyte antigen
(CLA) and the α4β7 integrin molecule, which mediate lymphocyte homing to the skin and to the gastrointestinal tract, respectively. The α4β1 integrin (also known as VLA-4) has less specificity than CLA and α4β7, in that it may direct lymphocytes to several organs. The homing integrins allow lymphocytes to bind to counter-receptors on the high endothelial venules resulting in a weak adhesion. CLA binds to E-selectin located on skin endothelium, whereas α4β7 binds mucosal addressin cell adhesion molecule 1
(MAdCAM-1) which is located on gut endothelium, and VLA-4 binds vascular cell adhesion molecule 1 (VCAM-1) which is expressed on inflamed endothelium at various
5 tissue sites [28]. The endothelial cells secrete chemokines, which allow a stronger adhesion by activating the integrins on the lymphocyte. These interactions then allow the lymphocytes to extravasate from the blood stream into specific organ sites [8]. Despite its lack of tissue specificity, studies in both animal models and humans suggest that α4β1 integrin plays a central role in lymphocyte recruitment to the lung. Animal models have shown that CD4+ effector memory T-cells express α4β7 in the gut and α4β1 in the lung tissue [31-35]. Human studies show that whereas peripheral blood CD4+ effector memory T-cells have heterogeneous expression of α4β1, α4β7 and CLA [28, 36], resident memory CD4+ T-cells of the lung predominantly express VLA-4, but are CLA-, and express only low levels of α4β7 integrin [30, 37].
Dendritic cells in the development of tissue-specific homing:
More recent studies have indicated that local populations of dendritic cells play a critical role in determining homing molecule expression on stimulated lymphocytes. Von Adrian et al purified DC from different lymphoid tissues of mice and incubated them in vitro with fluorescently labeled naïve T-cells. Stimulated T-cells were then injected into mice, and their localization evaluated with fluorescent microscopy. They observed that T-cells activated by DC isolated from intestinal lymphoid tissue expressed the α4β7 integrin and localized to the gut. In contrast, lymphocytes incubated with DC isolated from skin led to the expression of CLA and localized to the skin [38]. Mora et al subsequently determined that the homing properties of lymphocytes are elastic, as T-cells obtained from for one tissue can be re-trained to patrol another site. The investigators isolated
6 memory T-cells from specific tissues, washed these cells, and co-cultured them with DC from specific tissues. They found that, regardless of the tissue from which they had been initially isolated, the homing molecule expression and circulation patterns of T-cells could be altered to correspond to the tissue specificity of the last DC population they encountered [39].
The ability of DC to stimulate homing molecule expression appears to be dependent upon specific vitamins. Iwata et al showed that conversion of retinol A (Vitamin A) to retinoic acid by murine intestinal lymph node DC induces effector memory T-cells to express the
α4β7 integrin. They also found that mice that were starved of Vitamin A had a reduction in T-cells expressing α4β7 in the lymphoid organ and far fewer intestinal T-cells [40]. In contrast, DC isolated from the epidermis of sheep require Vitamin D to induce a skin- homing phenotype on stimulated T-cells, although this was based on induction of expression of the chemokine receptor CCR10 rather than the skin-specific homing molecule CLA [41].
Limitations of BCG vaccination:
Assessment of the protective efficacy of BCG has shown widely variable responses, from
80% in Northern Europe to 0% in India and Malawi [42]. A meta analysis of BCG efficacy surveyed 1264 titles or abstracts and selected 70 articles for in depth review. Of the 70 articles, 14 prospective trials and 12 case control studies met the criteria of having measured the efficacy of the BCG vaccination in preventing tuberculosis-related deaths.
7 They concluded that vaccination with BCG reduced the risk of tuberculosis by an average of 50% [43]. Many explanations have been offered for the lack of efficacy of BCG vaccination with regard to pulmonary tuberculosis. Some of these include the absence of specific immunogenic M. tuberculosis proteins in BCG [44], differences in prior environmental exposures of some populations to other mycobacteria [12, 42], the biological variability of the various BCG strains administered [45, 46], genetic differences between vaccinated populations [5], and the lack of clear dose optimization
[47]. However, given that BCG appears effective at preventing disseminated disease, but not at protecting against pulmonary tuberculosis, an additional possibility is that standard intradermal administration of BCG induces effective systemic immunity, but does not result in a recall response that can be optimally localized to the lung at the time of respiratory exposure to M. tuberculosis. Studies in mice have confirmed that the route of vaccination with BCG has a significant impact on homing molecule expression on memory T-cells capable of responding to M. tuberculosis [48]. Likewise, human studies comparing intradermal and oral administration of BCG have demonstrated that these routes of vaccination can skew the resulting memory T-cell responses towards expression of CLA or α4β7 integrin, respectively [49].
Assessment of local immunity in humans following respiratory exposure to M. tuberculosis:
In contrast to BCG vaccine recipients, individuals who have had previous respiratory exposure to M. tuberculosis are more clearly protected against subsequent re-infection
8 with the organism. Nevertheless, cells obtained from the lungs of such individuals using the technique of bronchoalveolar lavage (BAL) did not demonstrate clear differences from BAL cells obtained from PPD skin-test negative subjects in terms of percentage of lymphocytes present in BAL, or, more specifically, of numbers CD4+ T-cells in BAL.
We therefore theorized that the “protection” that these individuals had was a function of their ability to rapidly localize an antigen-specific immune response to the lungs at the time of re-exposure to M. tuberculosis. To model this re-exposure, we utilized the technique of bronchoscopic segmental antigen challenge. This method has previously been used extensively to characterize the local Th2-mediated immune responses in the lungs of atopic asthmatics using challenge with allergens such as ragweed [50-52]. We instead utilized PPD as the challenge antigen, and compared responses of naturally- infected skin-test positive subjects (i.e., those without a history of BCG vaccination) to responses of healthy PPD-negative controls. In our protocol, a baseline BAL sample was collected, after which 10 cc of normal saline was instilled as a control into the right middle lobe (RML), and 0.5 TU of PPD (1/10th of the standard skin-test dose) was instilled into the corresponding portion of the left lung, the lingula. Forty-eight hours later, BAL samples were obtained from both the control and challenged segments.
In our initial studies, we demonstrated that bronchoscopic challenge with PPD resulted in a localized lymphocytic inflammatory response in challenged lingular lung segments of
PPD-positive subjects (but not in the RML control segment). PPD-negative subjects displayed no response to the challenge. The cells recruited into the PPD-challenged segments of the skin-test positive segments were enriched for CD4+ T-cells, and for cells
9 that displayed antigen-specific production of IFNγ in response to in vitro stimulation with
PPD [53].
We subsequently examined the role of specific chemokines in the PPD-challenge response, and found that localized inflammation of skin-test positive individuals following bronchoscopic administration of PPD was associated with the production of
IFNγ-dependent CXCR3 ligands IP-10 and Mig, but not of CCR5 ligands MIP-1α and
RANTES. In contrast, skin-test negative subjects produced none of these chemokines.
This observation suggested that resident effector memory cells capable of early IFNγ production could be present within the lungs of naturally-infected PPD-positive subjects.
Further investigation confirmed that baseline BAL cells of PPD-positive subjects were greatly enriched for PPD-specific Th1-like cells. Furthermore, baseline BAL cells of skin-test positive subjects produced IP-10 and Mig in response to in vitro stimulation with PPD. These findings therefore suggested that localization of M. tuberculosis- specific cells to the lung at baseline played a central role in subsequent recruitment of additional antigen-specific cells to the lung as part of a protective recall response to the organism [54].
In the current study, we evaluated the role of homing molecule expression in the localization of PPD-specific recall responses to the lung. Our hypothesis was that the expression of the α4β1 integrin (VLA-4) plays a central role in localization of M. tuberculosis-specific Th1-like cells to the lungs of naturally-infected individuals. We evaluated expression of the α4β1 and α4β7 integrins as well as CLA in combination with
10 intracellular staining for PPD-specific production of IFNγ on CD4+ T-cells from peripheral blood, and from BAL obtained both before and after bronchoscopic challenge with PPD. We found that there is preferential recruitment of PPD-specific α4β1- expressing CD4+ T-cells to the lung after challenge. This suggests that expression of
α4β1 is required for optimal localization of M. tuberculosis-specific CD4+ T-cells to the lung. Our findings could be relevant to the development of future vaccine strategies, as current intradermal vaccination with BCG would be anticipated to induce development of a memory response in which expression of CLA is substantial, and in which localization of recall responses to the lung is therefore not optimized.
MATERIALS AND METHODS
Subjects:
Bronchoscopy subjects were healthy non-smoking volunteers aged, 22-46. All positive subjects had been found to have a positive tuberculosis skin-test previously (ranging from
1 to 10 years prior to their participation); none had a history of vaccination with BCG.
Of the PPD-positive subjects (composed of three males and one female), none had a history of active tuberculosis or of any symptoms suggestive of active tuberculosis, such as cough, night sweats, fevers, or weight loss. The exclusion criteria for these subjects included history of asthma or other chronic lung disease, history of cardiac disease, and history of adverse reactions to topical anesthetic agents. Negative subjects were
11 determined by a negative response to the tuberculin skin test. This group consisted of two males and one female.
All protocols involving human subjects were approved by the Institutional Board of
Review of Case Western Reserve University and University Hospitals of Cleveland and of the Louis Stokes Cleveland Department of Veterans’ Affairs Medical Center.
PPD:
Sterile commercially-prepared PPD (Tubersol®, Aventis Pasteur, Toronto) was used in skin-testing and bronchoscopic challenge procedures. To assure sterility, dilutions of
PPD were prepared in a laminar flow hood immediately prior to administration using previously unopened vials of Tubersol® and sterile physiologic saline.
PPD for in vitro use was obtained from the Staten Serum Institut (Copenhagen,
Denmark).
Confirmation of tuberculin skin testing status:
The self-reported PPD skin-test status of all subjects was confirmed by performing standard skin-testing in the laboratory. A dose of 5 TU (0.1 ml) of Tubersol® PPD was injected intradermally using 26G needles. All skin-test responses were measured by one of the investigators as mm of induration 48 hours after PPD administration. For the
12 purposes of this study, skin-test responses of 10 mm or more of induration were considered positive (with actual range 12 x 12 mm to 18 x 14 mm for the skin test positive subjects). Only subjects who displayed no induration in response to PPD were considered skin-test negative.
Isolation of peripheral blood mononuclear cells (PBMC):
Peripheral blood was obtained by venipuncture and mononuclear cells isolated by density sedimentation using Ficoll-Hypaque (Ficoll-Paque PLUS, Amersham Bioscience AB,
Uppsala, Sweden). Briefly, 15-20 ml of venous blood was diluted in 50 ml polypropylene tubes to a final volume of 35 ml with PBS (BioWhittaker, Walkersville,
MD). Fifteen ml of Ficoll-Hypaque was then under layered into the tubes using sterile glass pipettes, and samples were centrifuged at 1500 RPM (500 x g) for 45 minutes (with no brake). PBMC were collected from the Ficoll-serum interface using transfer pipettes and again placed into 50 ml polypropylene tubes. Cells were washed three times in
RPMI 1640 (BioWhittaker) and counted using a hemocytometer.
Bronchoscopy procedures:
All bronchoscopies were performed in the Case Western Reserve University General
Clinical Research Center (GCRC) at University Hospitals Case Medical Center. Pre- procedure lung and cardiac examinations were normal for all subjects. Before each procedure, subjects were anesthetitized with a nebulation treatment of 2% lidocaine
13 followed by gargling with 2% lidocaine. Topical anesthesia of the nasal passage was then performed using 2% viscous lidocaine. Further anesthesia was provided by topical application of 1% lidocaine to the airways via the bronchoscope. All subjects were observed in the GCRC for at least 30 minutes following each bronchoscopy procedure.
Subjects were provided the hospital pager number of one of the investigators and were advised to call if any symptoms of concern arose.
Bronchoscopy with bronchoalveolar lavage:
For procedures involving bronchoalveolar lavage only, the bronchoscope was wedged into one subsegment of the right middle lobe (RML). Lavage was performed by instilling six 30 cc aliquots of pre-warmed normal saline (NS) into the RML subsegment.
Following each instillation, lavage fluid was withdrawn under gentle suction.
Bronchoscopic segmental antigen challenge with PPD:
The procedure of bronchoscopic segmental antigen challenge with PPD is performed under the auspices of FDA IND BB-11182 has been described in detail previously [53]
Prior to participation, each subject underwent a screening evaluation that included a baseline chest x-ray (to exclude active tuberculosis) as well as a laboratory evaluation including complete blood count, comprehensive chemistry panel, and (for female subjects) urine pregnancy testing. The challenge protocol involved two bronchoscopy procedures. In the initial bronchoscopy, baseline bronchoalveolar lavage (BAL) of one
14 RML subsegment was performed with six 30 cc aliquots of NS, as described above. A control instillation of 10 cc of NS was then placed into a different subsegment of the
RML. The challenge dose of 0.5 TU of PPD was then instilled, in a volume of 10 cc NS, into a subsegment of the lingula. Subjects recorded their temperature as well as any symptoms observed for a period of seven days beginning the evening of the challenge procedure.
Repeat bronchoscopy was performed 48 hours after the challenge procedure. Prior to the follow-up procedure, subjects underwent a second chest x-ray and gave blood for repeat laboratory studies. Subjects were questioned regarding interval symptoms of cough, sputum production dyspnea, or chest pain, and examination of the heart and lungs was repeated. BAL of the saline control subsegment of the RML was then performed using six aliquots of 30 cc NS, after which BAL of the PPD-challenged subsegment of the lingula was performed, also using six 30 cc aliquots of NS.
Processing of BAL fluid:
BAL fluid from all procedures was placed on ice for transport to the laboratory. Fluid was aliquoted into 50 ml polypropylene tubes, and the total volume of BAL fluid recovered from each subsegment was recorded. Samples were then immediately centrifuged at 1500 RPM (480 x g) for 10 minutes. BAL cells were stained and counted using a hemocytometer.
15 Cytospin preparations were made using approximately 25-50,000 cells from each BAL sample. Resuspended BAL cells were placed in a slide centrifugation apparatus
(Cytofunnel, Shandon, Pittsburgh, PA) and centrifuged at 800 x g for 8 minutes in a
Shandon Cytospin 3 centrifuge. Slides were then stained with a rapid Wright-Giemsa stain method (LeukoStat, Fisher Diagnostics, Pittsburgh, PA). Cell differentials were determined by counting 300 cells from each sample under light microscopy.
Intracellular staining for IFNγ and assessment of BAL cells surface markers:
BAL cells were resuspended in IMDM (BioWhittaker) with 5% fresh autologous serum
(AS) and 1% penicillin G (Sigma, St. Louis, MO) at concentration 400-500,000/ml and aliquoted into 14 ml polypropylene tubes. Cells were then incubated with medium alone,
PPD (10µg/ml), or Staphylococcal Enterotoxin B (SEB) (1µg/ml) (Sigma), which was used as a positive control. After an initial 2-hour incubation at 37oC, 10µg/ml Brefeldin
A (#347688-BD Bioscience, San Diego, CA) was added to each tube. All samples were then further incubated at 37oC overnight for a total of 18 hours of stimulation.
The next morning, 100µl 20mM EDTA was added to each 1 ml sample. Following brief vortexing, samples were then incubated for 15 minutes at room temperature. Cells were then treated with FACS lysis buffer (#347691-BD Bioscience) and incubated for another
10 minutes at room temperature then wash buffer (1% bovine serum albumin/0.1% sodium azide in PBS) was added. Following centrifugation, at 1500 RPM (480 x g) for
10 minutes, cell pellets were resuspended in the wash buffer and aliquoted into
16 microcentrifuge tubes. These samples were then centrifuged at 2000 RPM (325 x g) and the cell pellet was used for surface staining.
BAL lymphocyte subsets were analyzed on samples incubated with medium alone by labeling of with fluorescent antibodies (all obtained from BD Bioscience). Antibody pairs were selected to allow for identification of CD4+ T-cells (CD3+/CD4+, using anti-
CD3-PE, #555340 anti-CD4-FITC, #340133), CD8+ T-cells (CD3+/CD8+, using anti-
CD3-PE as above, and anti-CD8-FITC, #347313), γδ T-cells (CD3+/TCRγd+, using anti-
CD3-PE and anti-TCR γd-FITC, #347903), and natural killer (NK) cells (CD3-/CD56+, using anti-CD3 FITC, #340542, and anti-CD56-PE, #347747). The lymphocyte gate was established by back-gating on CD3+ T-cells (from CD3-PE vs. forward-scatter plots).
Total numbers of lymphocyte populations present in each BAL sample were calculated by multiplying the percentage of specific populations present in the lymphocyte gate by the total number of lymphocytes (as determined by microscopy, above).
Homing molecule expression was evaluated on CD4+ T-cells of BAL cultured in medium alone, with PPD, and with SEB. Expression of the α4β1 integrin was assessed using anti anti-CD29 (β1)-PE (#12-0297-73-Ebioscience, San Diego, CA), anti-CD49d
(α4)-APC (#304308-Biolegend, San Diego, CA), and anti-CD4-PerCP (#347324-BD
Bioscience). To examine α4β7 we used anti-β7-PE (#555945-BD Bioscience), anti-
CD49d (α4)-APC (#304308-Biolegend), and anti-CD4-PerCP (#347324-BD Bioscience).
Expression of CLA was evaluated using used anti-CLA-FITC (#555947-BD Bioscience), and anti-CD4-PerCP (#347324- BD Bioscience). For all studies, samples of cells were
17 also incubated with appropriate IgG isotype control antibodies of each conjugate to establish proper gating for each antibody. Samples were vortexed, incubated in the dark for 10 minutes at room temperature and washed by addition of 1 ml of wash buffer followed by centrifugation at 2000 RPM (325 x g) for 10 minutes. Homing molecule expression on CD4+ T-cells was determined as the percentage cells displaying staining for each of the homing molecule expressed within the CD4+ gate. Total number of cells expressing a specific homing molecule was determined by multiplying the percentage of that specific homing population by the total number of CD4+ T-cells.
Intracellular staining for IFNγ was then performed. Cell samples were incubated for 10 minutes with 500µl of FACS permeability solution (#347691-BD Bioscience) at room temperature, washed with wash buffer, and then centrifuged at 2000 RPM (325 x g) for
10 minutes. After removal of supernatants, samples were incubated with 20µl of anti-
IFNγ-FITC (#340449, BD Bioscience) or, for some tubes, control antibody anti-IgG2a -
FITC (#11-4729-71-Ebioscience) and for the CLA stain anti-IFNγ-PE (#12-7319-71-
Ebioscience) in the dark for 30 minutes at room temperature. Samples were then washed with 1 ml wash buffer and centrifuged at 2000 RPM (325 x g) for 10 minutes.
Supernatants were removed and the labeled cells resuspended in 500µl of 1% paraformaldehyde and transferred to 5 ml polystyrene tubes. Samples were analyzed on a
3-laser flow cytometer (BD LSR, BD Bioscience) using FlowJo software (Treestar,
Ashland, OR). Homing molecule expression on CD4+ T-cells was determined by percent of each homing molecule expressed in the CD4+ gate that produced IFNγ. Total number of IFNγ producing cells expressing a specific homing molecule was determined by
18 multiplying the percentage of specific homing population producing IFNγ by the total number of CD4+ T-cells.
Evaluation of lymphocyte proliferation:
In order to clarify that increased numbers of CD4 lymphocytes in BAL 48 hours after
PPD challenge were observed because of cell recruitment, rather than proliferation, we used Ki-67 to assess in vivo and in vitro proliferative responses to PPD. Ki-67 is a human nuclear antigen that is present in proliferating cells and not in resting cells [55].
In vitro proliferation was assessed using PBMC and BAL cell samples obtained during the initial bronchoscopy procedure at baseline. Cells were resuspended at a density of
0.8x10^6 cells/ml in 10% AS and 1% penicillin G (Sigma), of which 1 ml was added to each well of 24-well plates. Baseline cells were compared to those incubated for 24, 48, and 72 hours with medium alone, PPD (10µg/ml), or SEB (4µg/ml). At each time point, cells were removed from each well and placed into 15 ml polypropylene tubes. Each well was then washed with IMDM (BioWhittaker) and 1% penicillin G (Sigma) to obtain any residual cells, which were also added to the appropriate tube. Each sample was then divided in half, transferred to eppendorf tubes, and spun down at 1500 RPM (480 x g) for
10 minutes. Surface staining was performed on the resulting cells pellets (approximately
0.4x106 cells per tube). For each condition, cells were incubated for 10 minutes with anti-CD3-APC (#17-0038-71-Ebioscience) and anti-CD4-PerCP (#347324-BD
Bioscience), or with the appropriate isotype controls. Cells were then washed with wash buffer and centrifuged again at 2000 RPM (325 x g) for 10 minutes. Intracellular staining
19 with Ki-67 was then performed. Each sample was incubated with 1 ml FACS lysis buffer
(#347691-BD Bioscience) for 10 minutes at room temp. Following another washing step, cells were permeabilized by incubation with 500µl FACS Perm (#347692-BD
Bioscience) for 10 minutes at room temperature. The cells were washed and centrifuged again at which point 20µl of anti-Ki-67-PE (#556027-BD Bioscience) was added to each sample. Following a 40 minute incubation at room temperature, samples were washed, centrifuged, and fixed by resuspension in 500µl 1% PFA and transferred to 5 ml polystyrene tubes.
In vivo proliferative responses to PPD were also assessed using BAL cells obtained during follow-up bronchoscopy 48 hours after bronchoscopic challenge with PPD. Ki-67 staining was performed in the manner described above on BAL cells from control (RML) and PPD-challenged (lingular) lung segments without further in vitro stimulation.
Statistical Analysis:
Comparisons involving multiple studies of the same subjects were performed using paired t-tests. All statistical analysis was performed using GraphPad Prism 3.0 software
(GraphPad Software, San Diego, CA)
20 RESULTS
a) Bronchoscopic challenge with PPD results in development of localized lymphocytic inflammation in challenged BAL segments:
As has been previously described, bronchoscopic challenge with PPD provides a model for local recall responses to M. tuberculosis, and more generally for the development of
Th1-like immunity within the human lung. As is illustrated in Figure 1, the technique involves advancing the bronchoscopic into a subsegmental bronchus of the right middle lobe (RML) to obtain a baseline bronchoalveolar lavage (BAL) sample. The bronchoscope is then moved into a different RML subsegment for the instillation of 10 cc of normal saline (NS), which serves as a control. The bronchoscope is then advanced into a subsegmental bronchus of the corresponding portion of the left lung, the lingula.
Bronchoscopic challenge is then performed by instilling 0.5 TU of PPD (in 10 cc of NS) into the lingular subsegment. Follow-up bronchoscopy is performed 48 hours later and consists of performance of BAL at the both the RML “control” subsegment and the lingular “challenged” segment.
As was observed in our previous studies, PPD challenge resulted in the development of a localized inflammatory response that was enriched for CD4+ BAL lymphocytes. Due to the small number of subjects in our study presented data is not statistically significant, unless noted. Figure 2 displays the mean numbers of total lymphocytes and of specific lymphocyte subsets present in baseline and 48 hours post-challenge BAL from three PPD
21 positive subjects. As illustrated, the mean number of total lymphocytes in post-challenge
BAL was 3.5 fold higher that that observed at baseline. Analysis of lymphocyte subsets by flow cytometry indicated an increase in the percentage of CD4+ T-cells from 61.7% at baseline to 66.9% following PPD challenge. There was a corresponding decrease in the percentage of CD8+ T-cells in BAL, from 31.5% at baseline to 24.9% after PPD challenge. Accordingly, the CD4 to CD8 ratio of BAL lymphocytes increased from 1.76 at baseline to 2.43 following challenge. In contrast, γδ T-cells and NK cells accounted for relatively low percentages of BAL lymphocytes both before and after challenge (1.3% to 0.8% for γδ+ T-cells and 5.4% to 7.2% for NK cells). Although no statistically significant for this number of subjects these findings are consistent with our previously published observation that bronchoscopic challenge of skin-test positive subjects with
PPD results in preferential recruitment of CD4+ T-cells to the lung 48 hours post challenge [53, 54].
b) Skewing of homing molecule expression on CD4+ T-cells in baseline BAL:
As a baseline measure of the importance of homing molecule expression in localization of CD4+ T-cells to the lung, we compared the expression of the α4β1 and α4β7 integrins and of CLA on peripheral blood and BAL CD4+ T-cells obtained prior to PPD challenge.
Unsorted PBMC and BAL cells were stimulated with PPD for 2 hours and then 18 hours with Brefeldin A, which blocks cytokine release from the cells. Surface marker analysis was then performed using fluorescent antibodies to simultaneously detect expression of
CD4 and of the homing molecules cutaneous lymphocyte antigen (CLA), which mediates
22 homing to the skin, the α4β7 integrin that preferentially directly lymphocytes to the intestinal mucosa, and the α4β1 integrin that has been reported to mediate recruitment of lymphocytes to the lung (in addition to other organs). The cells were then permeablized for intracellular staining with IFNγ to identify the PPD-specific cells. Figure 3 shows representative flow cytometry data from studies of a single PPD-positive subjects. Each plot has been gated to show results for lymphocytes that expressed CD4. Comparison of
CD4+ T-cells from PBMC (column 1) and from baseline BAL (column 2) indicates skewing of homing molecule expression, as the α4β1 integrin is expressed on a substantially higher percentage of BAL CD4 T-cells than on CD4 T-cells in peripheral blood. There is also an increased frequency of PPD specific Th1-like cells (IFNγ+) in
BAL (2nd column) compared to PBMC (1st column). Notably, the overwhelming majority of PPD-specific Th1-like cells in both baseline and post-challenge BAL express the α4β1 integrin. Flow cytometry plots such as these were used to derive our cumulative data.
Figure 4A shows the comparison of mean expression of homing molecules CD4+ T-cells in peripheral blood and BAL for four PPD-positive subjects. In PBMC, expression of the
α4β1 and α4β7 integrins were observed on similar proportions of CD4 T-cells (47.9% and 45.4%, respectively), whereas CLA expression was observed on 6.8% of these cells.
In contrast, 82.5% of baseline BAL CD4 T-cells expressed the α4β1 integrin, whereas the α4β7 integrin was expressed by only15.4% of these cells. Only 2.08% of BAL CD4+
T-cells expressed CLA. There was significant difference between baseline BAL and
PBMC in regards to α4β1 (p=0.0072) and α4β7 (p=0.0192). In comparison to PBMC,
23 therefore, baseline BAL CD4+ T-cells of PPD-positive subjects display enrichment for
α4β1 expression, indicating that trafficking of these cells from blood to the lung is selective.
To confirm that this finding was not unique to PPD-positive subjects, we also compared homing molecule expression on CD4+ T-cells in peripheral blood and baseline BAL of three skin-test negative controls (Figure 4B). As was observed for skin-test positive subjects expression of the α4β1 and α4β7 integrins were observed on similar proportions of CD4 T-cells in peripheral blood (49.6% and 42.3%, respectively), whereas CLA expression was observed on 7.9% of these cells. In contrast, 78.8% of baseline BAL
CD4 T-cells expressed the α4β1 integrin, whereas the α4β7 integrin was expressed by only 20.0% of these cells. Only 1.16% of BAL CD4+ T-cells expressed CLA. These results were significant for α4β1 (p=0.0021) and α4β7 (p=0.0331). Therefore PPD negative subjects, as well as PPD positive subjects, show skewing of homing molecules expression in baseline BAL CD4 T-cells compared to blood.
c) Role of homing molecule expression in recruitment of antigen-specific Th1 cells to the lung in response to bronchoscopic challenge with PPD:
We subsequently assessed homing molecule expression on BAL CD4+ T-cells following bronchoscopic challenge with PPD. The percentage of BAL CD4+ T-cells expressing the
α4β1 and α4β7 integrins, as well as CLA, before and after PPD challenge are displayed in Figure 5A (mean data for three PPD-positive subjects). Mild increases were observed
24 in the percentages of CD4+ T-cells expression of the α4β7 integrin (14.1% to 16.1%) and
CLA (1.8% to 4.7%) following bronchoscopic challenge with PPD. However, the α4β1 integrin remained the predominant homing molecule expressed on BAL CD4+ T-cells after PPD challenge, despite a decrease in percentage of CD4 T-cells expressing this integrin (from 84.0% of CD4+ T-cells in baseline BAL to 78.9% of post challenge CD4+
T-cells). The predominance of expression of the α4β1 on CD4+ T-cells recruited to the lung in response to PPD challenge is displayed more clearly when this data is displayed as the total numbers of BAL CD4+ T-cells that express each homing marker at baseline and 48 hours post challenge (Figure 5B). From baseline to 48 hours post challenge there was a 3.5 fold increase of α4β1-expressing CD4+ T-cells from 4.21x105 to 1.47x106. Of the CD4+ T-cells recruited to the lung post challenge, 76% express α4β1, whereas only
16% express α4β7 and 7.9% express CLA.
We also evaluated the impact of homing molecule expression on recruitment of PPD- specific Th1 cells to the lung. This data was calculated from flow cytometry results gated on CD4+ T-cells that displayed staining for IFNγ in response to in vitro stimulation with
PPD, as was presented for a representative subject in Figure 3. The percentages of PPD- specific BAL CD4+ T-cells expressing α4β1 integrin, α4β7 integrin, and CLA before and after PPD challenge are displayed for three PPD positive subjects in Figure 6A. As illustrated, expression of the α4β1integrin was observed on 75.6% of antigen-specific
Th1 cells in baseline BAL, and on 80.9% of PPD-specific Th1 cells from 48 hours post- challenge BAL. A corresponding decrease in α4β7 expression was observed, as 22.2% of PPD-specific Th1 cells in baseline BAL and 14.3% of those in post-challenge BAL
25 expressed this integrin. A modest increase in CLA expression on antigen-specific Th1 cells, from 2.1% in baseline BAL to 4.7% in post-challenge BAL was observed.
The role of the α4β1 integrin in recruitment of antigen-specific Th1 cells to the lung is displayed more dramatically when considered in terms of the total number of PPD- specific Th1 cells in baseline and post-challenge BAL (Figure 6B). This data was calculated by multiplying the total numbers of BAL CD4+ T-cells in each sample by the percentage of CD4+ T-cells that were positive both for IFNγ response to in vitro stimulation with PPD and for expression of specific homing molecules. We were able to express this data in terms of total PPD-specific Th1 cells present in BAL before and after
PPD challenge. As illustrated, the mean number of total PPD-specific CD4+ T-cells increased 3.2-fold following bronchoscopic challenge with PPD, from 2.7x104 to
8.8x104. The number of α4β1+ PPD specific CD4+ T-cells increased 3.3 fold from
2.1x104 to 7.0x104 following PPD challenge, therefore α4β1-expressing cells account for
80% of the PPD-specific cells Th1 cells recruited to the lung in response to bronchoscopic challenge. Given that only 47.9% of peripheral blood CD4+ T-cells expressed α4β1, this finding indicates preferential recruitment of α4β1+ cells to the lung as part of the recall responses to M. tuberculosis antigens.
d) Assessment of the role of lymphocyte proliferation in increasing BAL CD4+ T-cells in response to PPD challenge:
26 In order to confirm our assertion that increased numbers of CD4+ T-cells present in BAL after PPD challenge resulted primarily from lymphocyte recruitment rather than proliferation, we assessed proliferative responses to PPD both in vitro and in vivo using
Ki-67 staining. Baseline BAL and peripheral blood cells were stained with Ki-67 both prior as well as after stimulation 48 hours of in vitro stimulation with PPD. The 48 hour time point was chosen to allow comparison with the in vivo proliferative effects of PPD in the lung between the time of its bronchoscopic instillation and follow-up bronchscopy
48 hours later. As a positive control for proliferation, Ki-67 staining was also performed on both PBMC and baseline BAL cells following 72 hours of in vitro stimulation with
SEB. As illustrated in Figure 7A, the increase in Ki-67 uptake was minimal in PBMC stimulated in vitro for 48 hours with PPD, as staining for Ki-67 increased from 2.1% of
PBMC incubated with medium alone to 2.3% in the presence of PPD (1st row). Likewise, staining for Ki-67 was observed in 4.2% of baseline BAL cells incubated for 48 hours in medium alone and in 5.8% of BAL cells following 48 hours of stimulation with PPD (2nd row). These findings suggest that PPD induces very little lymphocyte proliferation within the 48 hour time frame of interest. In contrast, Ki-67 staining of PBMC and BAL cells following 72 hours of incubation displayed a much stronger proliferative response, as 68.9% of blood lymphocytes and 16.3% of lymphocytes in BAL displayed Ki-67 staining following this stimulus (Figure 7A, 3rd column).
Ki-67 staining of BAL cells obtained at baseline and 48 hours after PPD challenge, but without in vitro incubation, is displayed in Figure 7B. In vivo stimulation with PPD had no impact on BAL lymphocyte proliferation, as the percentage of CD4+ BAL T-cells
27 expression for Ki-67 was 6.1% for both baseline BAL and 48 hours after PPD challenge.
These findings indicate that the accumulation of Th1 cells in the lung in response to bronchoscopic administration of PPD could not be accounted for by lymphocyte proliferation, and therefore confirm our assumption that these cells were recruited to the lung in response to PPD challenge.
DISCUSSION
Tuberculosis remains a major international public health concern, and development of more effective tuberculosis vaccines is a priority of current tuberculosis research. The failure of the existing vaccine, the Bacillus of Calmette and Guerin (BCG) to clearly impact the incidence of pulmonary tuberculosis emphasizes the importance of clarifying mechanisms of local immunity within the lung to the pursuit of an improved vaccine.
Because individuals who have been infected with M. tuberculosis display relative protection from being re-infected with the organisms, we have studied local immunity in these subjects in order to better understand protective pulmonary recall responses to M. tuberculosis. In previous studies of tuberculin skin-test positive individuals who had not received BCG, we have demonstrated that bronchoscopic segmental antigen challenge using PPD induced a localized lymphocytic immune response that is enriched for antigen-specific CD4 Th1 cells. In contrast, skin-test negative subjects displayed no inflammatory response to bronchoscopic challenge with PPD [53]. We subsequently demonstrated that the IFNγ-dependent CXCR3 chemokine ligands IP-10 and Mig were
28 readily detectable within BAL fluid from skin-test positive subjects following PPD challenge, whereas MIP-1α and RANTES, both of which are ligands of CCR5, could not be detected in these subjects, and neither class of chemokines was observed in the BAL fluid of skin-test negative subjects in response to PPD. These findings led us to speculate that memory effector cells capable of early IFNγ production reside within the alveoli of naturally-infected PPD positive individuals. Subsequent studies confirmed that this was the case, and that the predominant source of early IFNγ production in these subjects were antigen-specific CD4+ T-cells [54].
In the current study, we sought to investigate the role of homing molecule expression in the recruitment of M. tuberculosis-specific Th1-like cells to the human lung as a component of local recall responses to M. tuberculosis. We demonstrated that, compared to peripheral blood, homing molecule expression within the lung is skewed at baseline toward expression of the α4β1 integrin (which we observed on 82.5% of CD4+ T-cells in baseline BAL in comparison to 47.9% of CD4+ T-cells in blood), and away from expression of the α4β7 integrin (present of only 15.4% of BAL compared to 45.4% of blood CD4+ T-cells). This finding was observed in comparison of blood and BAL CD4+
T-cells of both PPD-positive and PPD-negative volunteers. As reported previously, we found that bronchoscopic challenge with PPD led to an increase in both the total number of cells and the total number of lymphocytes present in challenged lung segments of
PPD-positive subjects. The ratio of CD4 to CD8 T-cells in BAL was increased following
PPD challenge as well. The skewing of homing molecule expression on BAL CD4+ T- cells was maintained following PPD-challenge, indicating that lymphocytes were
29 selectively mobilized from the periphery to the lung. Furthermore by combining labeling of cell surface markers with intracellular staining for IFNγ production in response to
PPD, we determined that 80% of antigen-specific Th1 cells recruited to the lung in response to PPD challenge expressed α4β1.
The accumulation of antigen-specific cells within the lung in response to PPD challenge could theoretically result from either the local proliferation of these cells or from their recruitment from the periphery to the lung. Ki-67 detects a nuclear antigen that is present only in proliferating cells and absent in resting cells [55]. To confirm that these observations reflected recruitment, rather than proliferation, of α4β1-expressing lymphocytes, we evaluated BAL lymphocyte proliferation with Ki-67 staining. We found no difference in Ki-67 uptake between the RML (control) and lingular (PPD- challenged) lung segments at 48 hours post challenge, or following 48 hours of in vitro stimulation of BAL or PBMC. Prior studies have shown the lung to be an anti- prolerative environment for lymphocytes due to effects of surfactant, products of alveolar macrophages, and pulmonary epithelial cells [56-58]. Our findings are consistent with previous studies by Seitzman et al, who showed that in a murine model of pulmonary immunity to sheep red blood cells, accumulation of lymphocytes does not require local T- cell proliferation and instead depends on recruitment using the technique of BrdU and
Thymidine H3 uptake by the cells [59]. The lack of early in situ proliferation in responses to PPD thus emphasizes the importance of lymphocyte recruitment in the early response to M. tuberculosis infection. Based on the predominance of α4β1 integrin on localization of lymphocytes to the lung, these findings also suggest that recruitment of M.
30 tuberculosis-specific recall responses to the human lung would benefit from an initial exposure that results in maximal expression of the α4β1 integrin on antigen-specific memory cells.
Our observations are consistent with previous studies in both animals and humans that have suggested that the α4β1 integrin is the predominant homing molecule involved in lymphocyte recruitment to the lung [32, 35, 37]. In both Th1 and Th2 associated diseases of the human lung, such as sarcoidosis and asthma, that involve lymphocyte accumulation, BAL CD4+ T-cells have been found to be enriched for expression of the
α4β1 integrin [33, 60]. Studies of sarcoidosis and asthma in humans have also shown that baseline BAL CD4+ T cells of these individuals express α4β1, as do the control subjects. This data is consistent with our results showing expression of the α4β1 integrin on BAL CD4+ T-cells of both PPD-positive individuals and PPD-negative individuals.
However, our study is the first to demonstrate the importance of expression of α4β1+
CD4+ T-cells to lymphocyte recruitment to the human lung as part of a presumably protective response to a specific respiratory pathogen.
The limited efficacy of BCG in preventing pulmonary tuberculosis emphasizes the importance of local immunity to M. tuberculosis in evaluation of tuberculosis vaccines.
As lymphocytes demonstrate preferential homing back to site of initial infection, the limitations of BCG may reflect the inability of the standard intradermal vaccination to stimulate an immune response that can be optimally localized to the lung in response to subsequent inhalation of M. tuberculosis. Our previous study indicated that BAL cells of
31 M. tuberculosis-infected individuals are greatly enriched for the presence of PPD-specific
Th1 cells that play a central role in subsequent lymphocyte recruitment via their contribution to production of IFNγ-dependent Th1-associated chemokines. Because of the skewing of homing molecule expression on BAL CD4+ T-cells both at baseline and in response to re-exposure to antigen, both the initial localization of memory cells to the lung and the ability of chemokine responses to recruit additional antigen-specific cells may be largely dependent upon the expression of the α4β1 integrin. One issue, therefore, may be that standard intradermal administration of BCG does not optimally promote
α4β1 expression on memory CD4+ T-cells, so that BCG-induced memory cells may not be preferentially localized to the lung. The issue of route of BCG administration and its impact of homing molecule expression has been a subject studies in both animal models and humans.
Assessment of the role of route of vaccination with BCG in animals has led to contradictory findings. A comparison of the protective effects of intradermal and respiratory delivery of BCG in guinea pigs indicated that the aerosolization of the vaccine induced higher local cellular immune responses and an improved protective effect [61].
Feng et al showed that when vaccination of mice with aerosolized H37Rv and BCG resulted in increased development of antigen-specific α4β1 CD4+ effector memory cells, as well as up-regulation of its conjugate vascular adhesion molecule VCAM-1 on respiratory endothelium. Subsequent aerosol challenge of these animals resulted in a significant and rapid expansion of α4β1 CD4+ effector memory cells within the lung.
Furthermore, treatment of the mice with anti-α4 antibodies lead a significant reduction in
32 lymphocyte recruitment to the lung in response to aerosolized M. tuberculosis [32].
Palendira et al examined the effects of multiple routes of vaccination by giving mice
BCG by intravenous, subcutaneous, and aerosol routes, followed by aerosol challenge with either BCG or M. tuberculosis. All of these routes induced development of some antigen-specific CD4+ T-cells that expressed α4β1, although aerosol vaccination resulted in the largest number of such cells. However, the investigators found that α4β1- expressing effector memory CD4 T-cells could eventually accumulate within the lungs of mice and control BCG or M. tuberculosis to similar degrees regardless of the initial route of vaccination [48]. However, this study did not examine the responses to the intradermal route of BCG administration, which is the current standard. In contrast,
Dorer et al showed when mice were vaccinated with oral BCG they showed an expansion of CD4+ population capable of secreting IFNγ upon PPD re-stimulation, but surprisingly these effector memory cells lacked α4β7 but expressed high β1 [62].
Hoft et al have performed numerous studies examining the effects of various methods of administration of BCG upon the resulting immune responses of human volunteers.
Intradermal vaccination with BCG induce a predominant mycobacterium-specific Th1 immune phenotype in peripheral blood [63], and subjects given an intradermal booster after the initial vaccination display greater antigen-specific lymphoproliferation and IFNγ production, as well as greater ability to limit mycobacterium growth in a variety of in vitro assays [64]. Hoft et al also showed that when humans were vaccinated with oral
BCG and given an intradermal booster, it induced inhibitory effects on DTH but increased mycobacteria-specific IFNγ responses that expressed α4β7 on PBMC. An
33 additional study examining homing molecule expression on antigen-specific cells showed that oral vaccination with BCG induced the development of antigen-specific memory cells that predominantly expressed α4β7, whereas following intradermal vaccination, antigen-specific cells were primarily CLA positive. The study made no assessment of expression of α4β1 integrin, however [49].
An ideal tuberculosis vaccine would be affordable, easily administered after birth, and be safe immunogenic and effective at all ages and in all populations, and should be effective at preventing both primary M. tuberculosis and re-infection [65]. It should also be appropriate for use on immunologically naïve individuals as well as the large world population that has already been vaccinated with BCG [66]. Many approaches are currently under investigation, including live attenuated vaccines, virus/bacteria vectored vaccines, DNA vaccines, subunit vaccines, booster vaccines as well as killed BCG and
Mycobacterium bovis combined with adjuvants [66]. Another option is that of exploring the routes of administration of BCG and different approaches can enhance local recall responses within the lung to improve protective immunity. Other vaccines have been given via mucosal routes to optimize their efficacy; these include live attenuated oral polo vaccine, oral rotavirus vaccine, live attenuated various oral cholera vaccines, nasal influenza vaccine and live attenuate oral typhoid vaccine [66]. This might be a better way to administer BCG or a new vaccine since M. tuberculosis is a mucosally transmitted pathogen. Aerosol delivery of BCG to humans has been explored and was found by
Rosenthal et al, to be well-tolerated and capable of inducing systemic immune responses
[67]. In follow-up of this study, another group utilized the aerosol-delivery protocol to
34 administer BCG to non-human primates, in which nebulized BCG was found to induce protection superior to intradermal vaccination [68]. The potential problem of administering nebulizer treatments to very young children (and in the underdeveloped world generally), is being addressed currently by Wong et al, who have described a means to spray-dry the BCG vaccine as an alternative to lyophilization. The resulting preparation may be amenable to aerosol delivery by simple inhalation [69].
A key component of development of the various proposed approaches to improving the effectiveness of M. tuberculosis vaccines will be evaluation of correlates of protective immunity, as direct assessment of the efficacy of multiple vaccines will not be feasible due to the need to study very large populations for extended periods of time. In our studies of local pulmonary recall responses to M. tuberculosis in naturally-infected individuals, we have established a basis for evaluating the limitations of current intradermal vaccination with BCG with regard to protection against initial infection with
M. tuberculosis. The concept of lymphocyte homing may be relevant both to the localization of sentinel memory cells within the lung and the recruitment of additional antigen-specific cells following respiratory exposure to the organism. Further assessment of these responses in individuals receiving BCG via intradermal as well as other routes, and in recipients of new candidate tuberculosis vaccines, may provide a uniquely relevant means to assess the potential of these approaches to improve vaccine efficacy against tuberculosis.
35 Figure 1: Technique of bronchoscopic segmental antigen challenge. Following performance of baseline bronchoalveolar lavage (BAL), 10cc of sterile saline (control, C) is instilled into a subsegmental bronchus of the right middle lobe. The antigen (Ag), 0.5 TU of PPD, is then instilled into a corresponding segment of the lingula. After 48 hours, repeat bronchoscopy with BAL of both the control and challenged segments is performed. (Off- label use of PPD is performed under the auspices of FDA Investigational Drug protocol BB-IND11182.)
36 70
60
10^5 50 BAL,
in 40 cells
of 30
Number 20
10
0 Baseline PPD-challenged
Segment
Figure 2: Lymphocyte subset recruitment in response to bronchoscopic challenge with PPD. Segmental challenge with PPD (0.5 TU) elicits lymphocytic alveolar inflammation. The results are displayed as total number of lymphocytes for each subset present in BAL. This number was calculated by multiplying the total number of BAL lymphocytes from each sample by the percentage of cells within the lymphocyte gate displaying the markers of each subset (as determined by flow cytometry). Mean and standard deviation of results from 3 positive subjects are illustrated. Total BAL lymphocytes increased 3.5-fold in response to PPD challenge. CD4+ T-cells accounted for 61.7% of lymphocytes in baseline BAL, and 66.9% of lymphocytes in post-challenge BAL.
Key: =total lymphocytes, =CD4+T-cells,
=CD8+T-cells =γδ Τ−cells, and =NK cells.
37 Peripheral blood Baseline BAL Post-challenge BAL
104 104 104
103 103 103 PE PE PE
4b1 2
2 4b1 2 4 1 10 4B1 10 10 FL2-H:
α β FL2-H: FL2-H:
1 10 101 101
0 0 0 10 10 10 0 1 2 3 4 10 10 10 10 10 100 101 102 103 104 100 101 102 103 104 FL1-H: IFNg FITC FL1-H: ifng FITC FL1-H: IGNg FITC 104 104 104
103 103 103 PE PE PE
4b7 2 2 4B7
4b7 2 α4β7 10 10 10 FL2-H: FL2-H: FL2-H:
1 10 101 101
0 0 0 10 10 10 0 1 2 3 4 10 10 10 10 10 100 101 102 103 104 100 101 102 103 104 FL1-H: IFNg FITC FL1-H: ifng FITC FL1-H: IGNg FITC 104 104 104
3 103 10 103
2 2 2 10 10 CLA 10 FL1-H: CLA FITC FL1-H: CLA FITC FL1-H: CLA FITC 1 1 101 10 10
0 0 0 10 10 10 0 1 2 3 4 100 101 102 103 104 10 10 10 10 10 100 101 102 103 104 FL2-H: IFNg PE FL2-H: IFN PE FL2-H: IFNg PE
Figure 3: Skewing of homing molecule expression on CD4+ T-cells from peripheral blood, baseline BAL and BAL obtained 48 hour after bronchoscopic challenge with PPD. Representative flow cytometry results of a single subject illustrate that the percentage of CD4+ T-cells expressing α4β1 integrin, α4β7 integrin, and CLA (y-axis) in peripheral blood (left) differ substantially from that observed in baseline BAL cells (center) and in BAL cells obtained 48 hours after PPD challenge (right). As illustrated, expression of the α4β1 and α4β7 integrins is relatively similar in peripheral blood, whereas CD4+ T-cells in both baseline and post-challenge BAL display marked skewing toward expression of the α4β1 integrin. Intracellular staining for IFNγ after overnight incubation with PPD (x-axis) also illustrates the increased frequency of PPD-specific Th1-like cells in BAL compared to peripheral blood both at baseline (center) and 48 hours post challenge (right), as previously described [54].
38 A. B.
100 100
* * 80 80
molecules total) molecules of 60 total) 60 homing of (% homing (% cells T specific 40 cells 40 T of specific CD4+ of on CD4+ * on 20 * 20 Expression
Expression
0 0 4b1 4b7 CLA a4b1 a4b7 CLA
Homing molecules Homing molecules
Figure 4: Baseline BAL of both PPD positive and negative subjects are enriched for CD4+ T-cells expressing the α4β1 integrin. 4A: α4β1 integrin is expressed on 82.5% of CD4+ T-cells in baseline BAL of PPD positive subjects, compared to expression on 47.9% of peripheral blood CD4+ T-cells. In contrast, only 15.4% of baseline BAL CD4+ T-cells express the α4β7 integrin, compared to 45.4% expression on peripheral blood CD4+ T-cells. CLA is expressed on only 2.08% of these BAL cells and 6.8% of PBMC. Data is displayed as mean and standard deviation of percentages of total CD4+ T-cells for 4 PPD positive subjects. 4B: α4β1 integrin is expressed on 78.8% of CD4+ T-cells in baseline BAL of PPD negative subjects, compared to expression on 49.6% of peripheral blood CD4+ T-cells. In contrast, only 20.0% of baseline BAL CD4+ T-cells express the α4β7 integrin, compared to 42.3% expression on peripheral blood CD4+ T-cells. CLA is expressed on only 1.1% of these BAL cells and 7.9% of PBMC. Data is displayed as mean and standard deviation of percentages of total CD4+ T-cells for 3 PPD negative subjects.
Key: =Baseline BAL, =PBMC, *=significance (p<0.05)
39 A. B.
100 4000
3500
80 )
10^3) 3000 molecules molecules x total of cells 2500 60 homing homing (%
(total 2000 cells T specific specific cells
40 T of of 1500 CD4+ on CD4+ 1000 on Expression 20 Expression
500
0 0 4b1 4b7 CLA total 4b1 4b7 CLA
Homing molecules Homing molecules
Figure 5: Predominance of α4β1integrin expression on BAL CD4+ T-cells recruited to the lung in response to bronchoscopic challenge with PPD. Homing molecule expression on CD4+ T-cells from baseline BAL and BAL obtained 48 hours after bronchoscopic challenge with PPD. 5A: The percentage of BAL CD4+ T-cells expressing the α4β1 decreased from 84% at basline to 78.9% in post-challenge BAL, whereas the percentage of α4β7 expressing cells increased from 14.1% to 16.1%. and CLA expression increased from 1.8% to 4.7%. Data is displayed as mean and standard deviation of percentages of total CD4+ T-cells for 3 PPD postive subjects. 5B: Total numbers of BAL CD4+ T-cells expressing specific homing molecules at baseline and 48 hours following PPD challenge. When expressed as a percent of total increase in numbers of CD4+ T-cells recruited to the lung, 76% of recruited BAL CD4 T-cells expressed the α4β1 integrin, 16% expressed α4β7, and 7.9% expressed CLA. Data is displayed as mean and standard deviation of total number of CD4+ T-cells for 3 PPD postive subjects.
Key: =Baseline BAL, =48 hours post challenge BAL
40 A. B.
100 140 on
10^3) 120 x 80 total) cells molecules
of 100 molecules (%
60 (total 80 homing cells homing cells T T 60 CD4+ 40 specific specific CD4+ of of 40
20 PPD-specific Expression
Expression 20 on PPD-specific
0 0 4b1 4b7 CLA total 4b1 4b7 CLA Homing molecules Homing molecules
Figure 6: PPD-specific Th1-like CD4+ T-cells recruited to the lung in response to bronchoscopic challenge overwhelmingly display expression of the α4β1 integrin. Antigen-specific cells were identified by intracellular staining for IFNγ in response to overnight incubation with PPD (10µg/ml) in vitro. Comparison was made of homing molecule expression on antigen-specific Th1 cells from baseline BAL and from BAL obtained 48 hours after bronchoscopic challenge with PPD. 6A: The percentage of antigen-specific Th1 cells that expressed the α4β1 increased from 75.6% at baseline to 80.9% in post-challenge BAL, whereas the percentage of α4β7 expressing cells decreased from 22.1% to 14.3%. CLA expression increased from 2.1% to 4.7% of CD4+ T-cells in post-challenge BAL. Data is displayed as mean and standard deviation of percentages of total antigen specific CD4+ T-cells from 3 PPD positive subjects. 6B: Total numbers of PPD- specific Th1 cells in BAL at baseline and following bronchoscopic challenge with PPD. CD4 T-cells expressing the α4β1 integrin accounted for 80% of antigen-specific Th1 cells recruited to the lung in response to PPD challenge, whereas 14% expressed the α4β7 integrin and only 6% expressed CLA. Data is shown as mean and standard deviation of total number of PPD specific CD4+ T-cells from studies of 3 PPD-positive subjects.
Key: =Baseline BAL, =48 hours post challenge BAL
41 A. Medium (48hrs) PPD (48hrs) SEB (72 hr)
100 100 100
80 80 80
97.8 2.16 97.7 2.31 31.1 68.9 60 60 60 % of Max % of Max % of Max PBMC 40 40 40
20 20 20
0 0 0 0 1 2 3 4 100 101 102 103 104 100 101 102 103 104 10 10 10 10 10 Ki-67-PE Ki-67-PE Ki-67-PE 100 100 100
80 80 80
95.8 4.25 94.1 5.88 83.7 16.3 60 60 60
BAL % of Max % of Max % of Max 40 40 40
20 20 20
0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 Ki-67-PE Ki-67-PE Ki-67-PE
B. Baseline BAL Post-challenge BAL
100 100
80 80
93.8 6.17 93.9 6.11 60 60 % of Max % of Max 40 40
20 20
0 0 0 1 2 3 4 0 1 2 3 4 10 10 Ki-67-PE10 10 10 10 10 Ki-67-PE10 10 10
Figure 7: CD4+ T-cells do not show significant proliferative responses to 48 hours of stimulation with PPD. Proliferative responses to both in vitro and in vivo stimulation with PPD stimulation were assessed using Ki-67 intracellular staining. For each figure, shaded plots indicate results for samples incubated with isotype control antibody, whereas overlaid non-shaded plots indicate samples stained with Ki-67. 7A: Baseline BAL and PBMC were stimulated with PPD in vitro for 24 and 48 and with SEB for 72 hours as a positive control. Ki-67 straining of CD4+ T-cells is displayed following 48 hours of incubation in medium alone (1st column) and in the presence of PPD (10µg/ml, 2nd column), and illustrate the minimal effects of PPD on lymphocyte proliferation at this early time point. In contrast, substantial staining for Ki-67 is observed in CD4+ T-cells from both PBMC and BAL following 72 hours of incubation with SEB (3rd column). 7B: To assess the impact of bronchoscopic PPD challenge on in vivo lymphocyte proliferation, BAL cells obtained at baseline (left) and 48 hours after bronchoscopic challenge with PPD (right) were were stained with Ki-67 immediately following the bronchoscopy procedures. PPD challenge had no impact on Ki-67 staining of these two samples, as shown.
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49