The End of the Binary Era: Revisiting the

Spectrum of Tuberculosis
This information is current as
of October 22, 2018.
Philana Ling Lin and JoAnne L. Flynn
J Immunol 2018; 201:2541-2548; ;
doi: 10.4049/jimmunol.1800993
http://www.jimmunol.org/content/201/9/2541

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The End of the Binary Era: Revisiting the Spectrum of
Tuberculosis
Philana Ling Lin* and JoAnne L. Flynn†
Human Mycobacterium tuberculosis infection was
thought to result in either active symptomatic tuber-
culosis (TB) or latent asymptomatic infection. It is
now clear that this binary classification is insufficient
to describe the myriad of infection outcomes. In active
TB, symptomatic disease can be mild to severe, with a
range of lung and thoracic lymph node involvement or
extrapulmonary manifestations. Most humans control
the infection and develop latent TB infection, with
differential risks of reactivation to active TB. However,
some frequently exposed persons appear to be resistant
to infection, whereas others may initially become in-
fected yet subsequently eliminate all bacilli. The im-
munologic factors influencing these varied outcomes
are still not clear, but likely involve a range of different
responses. In this article, we review the data supporting
the spectrum of M. tuberculosis infection in humans as
well as data in nonhuman primates that allow dissec-
tion of the immune responses leading to the varied
outcomes of infection. The Journal of Immunology,
2018, 201: 2541–2548.
T
uberculosis (TB) remains a major cause of morbidity
and mortality worldwide and is now the most com-
mon cause of death from an infectious disease, sur-
passing HIV/AIDS (1). Even with improved measures to
diagnose and treat TB, the disease continues to ravage de-
veloping countries, causing 1.5 million deaths in 2016 (2).
Standard therapy for TB has not changed in almost 50 years
and involves 6 mo of treatment (four drugs for 2 mo, fol-
lowed by two drugs for an additional 4 mo). This long course
of treatment can result in poor compliance and the potential
for developing drug resistance. Drug-resistant TB is compli-
cated to manage and requires longer treatment with less ef-
fective drugs and greater risk of adverse effects. Although there
are newer drugs being used for drug-resistant TB, an ef-
fective vaccine that prevents infection or disease is essen-
tial for ending the scourge of TB. However, identifying
the protective mechanisms necessary for an effective TB
*Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of
Pittsburgh Medical Center, University of Pittsburgh School of Medicine, Pittsburgh,
PA 15224; and †Department of Microbiology and Molecular Genetics, University of
Pittsburgh School of Medicine, Pittsburgh, PA 15261
ORCIDs: 0000-0001-5169-3714 (P.L.L.); 0000-0001-7874-8981 (J.L.F.).
Received for publication July 17, 2018. Accepted for publication July 28, 2018.
This work was supported by the Bill and Melinda Gates Foundation (to J.L.F. and P.L.L.),
Aeras (to J.L.F.), and National Institutes of Health Grants AI111871 and AI134195 (to
P.L.L.) and HL110811, AI114674, AI123093, and AI094745 (to J.L.F.).
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800993
vaccine, or even correlates of protective immunity, has
proved difficult.
Mycobacterium tuberculosis, the bacterium that causes TB, is
an intracellular pathogen transmitted in aerosolized droplets,
typically by coughing from a person with active TB disease. In
the airways, the bacillus primarily infects alveolar macro-
phages but can infect other cell types. It then transits to the
lung parenchyma where it can infect resident macrophages
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or other phagocytes, including neutrophils. The ability to
replicate in macrophages is critical to the pathogenesis of
M. tuberculosis. Immune signals produced by infected mac-
rophages and bacterial products recruit other immune cells,
including monocytes, which differentiate into macrophages
and provide additional targets for infection. Simultaneously,
the bacilli can be taken up by dendritic cells and transported
to the thoracic lymph nodes where T cells are primed against
a broad range of M. tuberculosis Ags. These T cells return to
the site of infection in the lung and organize around infected
macrophages to form granulomas, the pathologic structure
most closely associated with TB (Fig. 1). Granulomas also
form in thoracic lymph nodes where they serve as a reservoir
of infection and dissemination for M. tuberculosis.
The course of M. tuberculosis infection is extremely variable
in humans. Once infection is established, most people control
but do not eliminate the initial infection. Infected but
asymptomatic individuals are classified as having latent
M. tuberculosis infection (LTBI). Persons with LTBI are
characterized as having evidence of infection by an immu-
nologic test [positive tuberculin skin test (TST) or IFN-g
release assay (IGRA)], with no signs or symptoms of TB
and are presumed to be noncontagious. A small percentage
(5–15%) of these people progress to active, symptomatic, and
transmissible TB within 2 y of infection, likely representing a
lack of initial control of infection; this is termed primary TB.
Symptoms of disease evolve slowly, possibly due to the slow
growth of the M. tuberculosis. Not surprisingly, active TB can
present in a variety of ways, most commonly as pulmonary
TB, but the bacillus can infect any organ in the body.
Pulmonary TB can present with mild, moderate, or severe
respiratory and systemic symptoms, with involvement of
Address correspondence and reprint requests to Dr. JoAnne L. Flynn, Department of
Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine,
Room 5058 Biomedical Science Tower 3, 3501 Fifth Avenue, Pittsburgh, PA 15261.
E-mail address: joanne@pitt.edu
Abbreviations used in this article: BCG, bacille Calmette–Guérin; CT, computed to-
mography; FDG, 2-deoxy-2-[18F]-fluoro-D glucose; IGRA, IFN-g release assay; LTBI,
latent M. tuberculosis infection; PET, positron emission tomography; TB, tuberculosis;
TST, tuberculin skin test.
Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$37.50
2542
FIGURE 1. Caseous granuloma from the lung of an M. tuberculosis–infected
macaque. Original magnification 34.
single or multiple lung lobes. Cavitary TB is seen in some, but
not all, individuals with pulmonary TB. Active TB can also
occur by reactivation of LTBI, resulting in a similar range of
severity as primary TB. It is estimated that there is a 10%
lifetime risk of reactivation in those with LTBI (3). However,
the more recent paradigm of LTBI (4) as a spectrum of in-
fection suggests that this risk is not the same for each person
with LTBI. The factors defining this reactivation risk are
unclear and likely involve both microbiologic and immuno-
logic components.
Diagnosis of M. tuberculosis infection is indirect and is
based on detecting cellular immune responses against myco-
bacterial proteins, either by a positive TST or IGRA. The
TST measures a delayed-type hypersensitivity reaction to the
injection of a poorly defined mixture of mycobacterial Ags,
known as purified protein derivative, but can yield false
positive results from cross-reactivity with nontuberculous
Mycobacterium spp. as well as M. bovis. More recently, IGRA
blood tests have been used, which rely on measuring IFN-g
produced by T cells ex vivo in response to the M. tuberculosis–
specific Ags ESAT-6 and CFP-10. These Ags are absent from
the commonly used TB vaccine bacille Calmette–Guérin
(BCG), an attenuated strain of M. bovis, and thus IGRAs can
distinguish reactions to M. tuberculosis from those to BCG.
However, these Ags are present in certain other mycobacteria,
such as M. africanum. Thus, although this is an improvement
over purified protein derivative skin testing, IGRAs remain an
imperfect test for M. tuberculosis infection. Importantly, nei-
ther IGRA or TST differentiates active TB versus LTBI, and
active TB must be diagnosed by the presence of clinical signs,
radiography, sputum smear for acid-fast bacilli, or culture for
M. tuberculosis.
The spectrum of TB in humans
The dogma of the binary nature of M. tuberculosis infection
(active TB versus LTBI) is an oversimplified and now out-
dated concept. M. tuberculosis infection has a spectrum of
manifestations (5–7) that encompass a broad range of out-
comes, including resisters (no evidence of infection despite
repeated exposure to M. tuberculosis); infected initially but
able to eradicate M. tuberculosis; infected but asymptomatic
and stable; latently infected but at high risk of reactivation;
active TB with chronic symptoms; and fulminant, severe tu-
berculous disease (Fig. 2). This concept has become better
BRIEF REVIEWS: SPECTRUM OF M. TUBERCULOSIS INFECTION
appreciated over the last decade owing to technologic ad-
vances in transcriptional profiling, in vivo imaging, and more
innovative and comprehensive clinical research that has en-
abled us to gain more thorough insights into TB pathogenesis.
The expanding definition of LTBI: resisters and reverters. A growing
appreciation for the complexities of M. tuberculosis exposure
and infection has developed from household contact studies
in which certain individuals, despite multiple exposures to an
index case, have persistently negative TST or IGRA results
and appear to be resistant to M. tuberculosis infection. These
individuals are termed “resisters.” This phenomenon is de-
scribed in some of the older literature and was nicely dem-
onstrated in a detailed and extended contact-tracing study
involving a single TB index case aboard a naval vessel
(known as the Byrd study) (8). In that study, despite
similar and prolonged exposure to the index case, several
subjects remained TST-negative, whereas many others
became TST-positive (8). Resisters were initially thought to
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be subjects who were not as frequently or severely exposed to
the index case. However, household contact investigation
studies have since identified subjects with no evidence of
infection (9) as long as 2 y after index case exposure
(10, 11). This is a fascinating cohort of people from which
clues about resistance to infection can be gleaned; this resister
phenotype has been better defined recently (reviewed in Ref.
12). It should be noted that the current diagnostics for
M. tuberculosis infection are relatively poor at identifying this
resister population. The TST can be cross-reactive to other
Mycobacterial spp., and the IGRA depends on detecting only
one immune response to M. tuberculosis infection (IFN-g
produced in response to two Ags). Thus, it is possible
that some IGRA-negative subjects may, in fact, have been
infected but are controlling (or eliminating) the infection by
IFN-g–independent mechanisms or have T cells that recognize
Ags other than ESAT-6 and CFP-10. Functional Ab
responses have been shown recently to distinguish LTBI from
active TB cases (13), but there are no data yet on Abs in resister
cohorts. Other immune mechanisms, such as nonclassical
T cells (e.g., gd T cells or mucosal-associated invariant
T cells) or innate immune functions of alveolar macrophages,
could be responsible for the apparent lack of infection observed
in this population (14). In any event, this special group of
people provide a unique opportunity for more detailed
immunologic and transcriptomic studies to determine the
mechanisms of protection against infection or disease.
In addition to resisters, another group of interesting subjects
has been recently identified: household contacts of an index
case or those who live in a high endemic area who develop
a positive IGRA but then revert to negative within 2 y
(“reverters”) (15–17). Some cases of reversion likely represent
the imprecise nature of the cut-off between positive and
negative IGRA results, given the marginally positive IFN-g
levels often observed. However, a substantial number of pa-
tients with high IFN-g responses truly reverted to IGRA-
negative within 2 y. This unique subset of patients may
represent initial infection with subsequent self-cure or eradi-
cation. A recent vaccine trial in humans found that revacci-
nation with BCG or with the H4 subunit vaccine of
mycobacterial proteins did not significantly reduce initial
infection and IGRA conversion but instead reduced the rate
of sustained IGRA conversion over the 2-y follow-up (18).
The Journal of Immunology 2543

FIGURE 2. The outcome of M. tuberculosis infection includes a host of outcomes and clinical manifestations. Those with severe outcomes are often highly
symptomatic (e.g., weight loss, chills, night sweats, fevers, cough) with a positive skin test or IGRA, chest x-ray (CXR) with substantial TB disease, and growth of
M. tuberculosis on sputum culture. In contrast, some individuals may have been infected in the past (with or without a positive IGRA/skin test) and have cleared
the infection resulting in the absence of symptoms, negative sputum culture, and normal chest x-ray. Although the greatest proportion of infected individuals are

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able to control infection (LTBI), the exact distribution of outcomes depicted in this article is not known. PPD, purified protein derivative TST.

BCG revaccination of previously BCG vaccinated subjects
resulted in reversion of an initially positive IGRA with an
efficacy of 45%, suggesting these vaccines had a robust re-
duction, or possibly elimination, of an initial M. tuberculosis
infection. As with resisters, the biologic mechanisms leading
to this outcome are of interest for development of preventive
strategies and provide new outcome measures for vaccine
trials. Additional studies to identify the true infection status
and more detailed analysis of immunologic responses of these
reverters are critical to more fully understand the spectrum of
LTBI. It is possible that there is a link between resisters and
reverters, with common immunologic mechanisms responsi-
ble for preventing or eliminating infection, respectively.
Subclinical disease. Subclinical TB, often described as having
detectable M. tuberculosis in sputum but with a normal chest
x-ray and no symptoms of TB, has been documented
in immune competent individuals. However, it is likely
underreported, because sputum examinations are not
typically performed on patients without symptoms or with
negative chest radiographs (8, 19). In the aforementioned
Byrd study (8), one of the exposed subjects was found to be
sputum positive with a normal chest radiograph and no overt
symptoms. Only after intensive questioning did this
individual admit to having 6 wk of cough and weight loss,
which resolved (8). Surprisingly, recent studies identified
some patients who would have been defined as having LTBI
but had a positive sputum sample, performed serendipitously
for research purposes (19). Similarly, features of active TB
have been historically observed at autopsy of patients who
died of other non-TB causes, suggesting that these patients
were otherwise asymptomatic of TB (20). With the
resurgence of TB during the HIV epidemic, clinicians have
recognized that immunodeficiency is often associated with
atypical clinical and radiographic manifestations of TB.
HIV-infected patients, especially those with low CD4
counts, do not generally have cavity formation and can have
atypical or even normal chest radiographs despite having
confirmed disease (reviewed in Refs. 21 and 22). Patients
on TNF inhibitors, a major risk factor for reactivation of
LTBI, also can have atypical manifestations of TB with
more disseminated disease (23). The quality of chest x-rays
has greatly improved from the time they were first used more
than five decades ago and this has led to improved diagnosis
of TB. More sophisticated imaging technologies such as
computed tomography (CT) have been used in research and
specific clinical settings, resulting in even more sensitive
detection of disease. Positron emission tomography (PET),
using 2-deoxy-2-[18F]-fluoro-D glucose (FDG, a PET probe
measuring metabolic activity), combined with CT has
enabled researchers and clinicians to find metabolically active
granulomas in otherwise asymptomatic or LTBI patients. In
fact, a spectrum of image patterns has been observed in subjects
with clinically defined LTBI (24) and in patients with
symptomatic active disease (reviewed in Ref. 25). Esmail
et al. (26) reported on 35 HIV-infected, antiretroviral naive
patients without signs or symptoms of active TB and identified
10 patients with PET/CT characteristics of active or subclinical
disease (e.g., infiltrates or metabolically active nodules) and
who also exhibited a higher risk of progression to active TB.
In this same study, there were 195 HIV+ patients who were
screened for TB but who were excluded from the study for
various reasons. Of these 195 patients, 10 were found to be
sputum positive, of whom five were asymptomatic with normal
chest x-rays. Clearly, there is discordance between the
traditional classification of LTBI and active TB.
Transcriptional studies of human blood have shown over-
lapping signatures between clinically defined active TB and
LBTI (19, 27) as well as between LTBI and uninfected sub-
jects (28). These studies support the existence of a full spec-
trum of M. tuberculosis infection beyond the dogmatic,
clinically defined, binary outcome. Recent blood transcrip-
tional studies identified a signature that could predict active
TB as early as 18 mo prior to clinical diagnosis (29). Although
these studies do not provide biological mechanisms, the pre-
dictive signatures reflect ongoing disease evolution that cannot
otherwise be detected by clinical signs or symptoms. To ac-
count for this broader classification of M. tuberculosis infec-
tion, some have coined the term “incipient TB.” Although
this term has been disputed since the early 1900s (30), it is
now defined by the World Health Organization as “prolonged
2544 BRIEF REVIEWS: SPECTRUM OF M. TUBERCULOSIS INFECTION
asymptomatic phase of early disease during which pathology
evolves, prior to clinical presentation as active disease” (31).
Tests that can identify this phase of infection will facilitate
more strategic treatment of large populations, preferably by
identifying and treating those at greatest risk of active TB
before they become overtly contagious, thereby reducing
transmission. To more fully understand the spectrum of
M. tuberculosis infection and the underlying mechanisms that
drive outcomes (especially with regard to incipient infection),
more in-depth studies of basic TB pathogenesis and biology
of the host and pathogen are required.
Animal models for understanding the spectrum
of M. tuberculosis infection
The human data that support the idea of a spectrum of
M. tuberculosis infection outcomes are compelling. However,
for practical reasons, much of this data comes from blood,
whereas TB is a disease primarily of the lungs. Animal models
have contributed greatly to our understanding of the key
events during infection that dictate outcome and allow one
to control the infection dose and timing, to perform inten-
sive monitoring and serial sampling, and to harvest tissue
samples, including granulomas, lung, and lymph nodes, at
various timepoints postinfection. Although murine models
are generally used to study pathogenesis and immunology of
TB, mice do not replicate many key aspects of human
M. tuberculosis infection, including granuloma formation and
LTBI. For these reasons, the available data on LBTI and in-
dividual granulomas are primarily from nonhuman primate
models. Macaques are unique models for TB in that they
recapitulate the entire human range of infection outcomes and
exhibit pathology, including granulomas, that is identical to
human M. tuberculosis infection. In particular, cynomolgus
macaques infected with a low dose of virulent M. tuberculosis
develop the full spectrum of M. tuberculosis infections seen in
humans, from LTBI to severe disease, over the course of
several months (32, 33). Rhesus macaques are more suscep-
tible, with most developing active TB within 4 mo; however,
even in rhesus macaques, there is a spectrum of active TB
from mild to severe (34). Thus, these macaque models pro-
vide unique opportunities to investigate M. tuberculosis host–
pathogen interactions with remarkable similarities to humans.
Early events in M. tuberculosis infection
In humans, it is extremely difficult to study early events of
infection, because the time of infection is rarely known and the
diagnostics rely on development of an adaptive immune re-
sponse, which usually takes 6–8 wk to be detected by IGRA or
TST. However, the available data indicate that early immune
control of M. tuberculosis infection plays a critical role in
outcome (35). As an intracellular respiratory pathogen, the
bacilli first encounter the immune response in the airways,
which includes cellular (e.g., alveolar macrophages, T cells,
innate lymphocytes) and noncellular (e.g., antimicrobial
peptides) (36) components. Alveolar macrophages are capable
of killing M. tuberculosis soon postinfection, but these cells are
quite heterogeneous in both humans and macaques, with
likely variable bactericidal capacity. Specific subsets of alveolar
macrophages observed before M. tuberculosis infection were
associated with differences in infection outcome (active ver-
sus LTBI) (37). Infected macrophages can induce bronchial
epithelial cells to express DEFB4 and other antimicrobial
effectors that can kill M. tuberculosis directly (36). These very
early events are consistent with our findings in which serial
PET/CT imaging was performed during M. tuberculosis in-
fection in macaques to determine whether the pattern of lung
granulomas could predict outcome. Animals that would later
develop active TB had more granulomas as early as 3 wk
postinfection (before the adaptive immune response is estab-
lished) with significantly more new granulomas developing
because of dissemination from existing granulomas between
3–6 wk compared with animals that would later present with
LTBI (38). These data suggest that innate and early adaptive
responses are critical to outcome.
Bacterial burden within individual granulomas peaks at
∼4 wk postinfection when there is minimal bacterial killing
detected (39). Substantial bacterial killing occurs within
granulomas by 10–11 wk postinfection, coinciding with the
development of an adaptive immune response. RNA tran-
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scriptional profiling of granulomas at 4 wk shows a pre-
dominantly proinflammatory profile that is later reduced by
12 wk postinfection (40). Transcriptional signatures in blood
from macaques after M. tuberculosis infection appear to be
similar to human signatures (41–43). Interestingly, some of
the signatures (e.g., IFN-related signatures) that predicted
active TB during the period of clinical stability in hu-
mans (so-called incipient TB) (29) were observed prior to
M. tuberculosis infection in our macaque model (41). In a
rhesus study of a CMV vector-based vaccine, Hansen et al.
(42) observed upregulation of a transcriptional module
related to neutrophil and innate immune function associ-
ated with vaccine-induced protection that was present be-
fore infection. These data reinforce the notion that early
immune factors are critical determinants of outcome.
The success and failure of the granuloma
The histopathologic hallmark of TB is the granuloma, an
organized spherical structure composed of a variety of cell
types, primarily macrophages and lymphocytes. The classic
granuloma has a necrotic (caseous) core, surrounded by epi-
thelioid macrophages, with an outer cuff of lymphocytes and
macrophages. However, there is a range of granuloma types,
including nonnecrotic, suppurative (neutrophilic), fibrotic,
and mineralized. These diverse granuloma types were recog-
nized early in studies on human TB, and we observe the full
range of granuloma types in macaques (44). The granuloma
contains and prevents dissemination of the infection, and it
provides an immune microenvironment where macrophages
are activated to kill or restrain the growth of M. tuberculosis
bacilli. However, the bacterium has evolved to persist in some
granulomas, often for years or even the lifetime of the host.
The actual metabolic state of the bacilli in granulomas over
the long term is not known, although there likely is replica-
tion occurring, even if quite slowly or intermittently. What is
becoming clear is that there is substantial heterogeneity of
granulomas, even within the same host, in terms of host
response, bacterial growth and killing, dissemination, and
reactivation. This is exemplified by serial PET/CT imaging of
individual granulomas in which individual granulomas appear
dynamic and independent, increasing or decreasing in size
(measured by CT) and metabolic activity (as measured by
FDG avidity) within the same macaque and even within the
The Journal of Immunology
same lung lobe (38, 45) (Fig. 3). Even in humans, reso-
lution of some granulomas and progression of others dur-
ing active TB can be observed by PET/CT imaging over as
little as 2 mo (46).
We have shown that individual bacilli that enter the lung
give rise to individual granulomas, but these granulomas can
have different trajectories (39). The heterogeneity among
individual granulomas within the same host can be observed
in terms of bacterial burden and killing, immune responses,
risk of dissemination, and granuloma type (39, 44, 47, 48).
Using a library of “bar-coded” M. tuberculosis, in which in-
dividual bacteria can be distinguished by a DNA tag, in
conjunction with serial PET/CT imaging, we demonstrated
that only a subset of granulomas within a single host dis-
seminate to form new granulomas and advance disease (48).
Thus, despite the global host immune response to the path-
ogen, it is the local host–pathogen response in each granu-
loma that determines overall control of the infection. During
the first 4 wk of M. tuberculosis infection when adaptive im-
munity is just being initiated, bacterial burden in granulomas
is at its greatest with little to no bacterial killing (39). During
the course of infection, granulomas from animals with active
or latent disease can sterilize the infection, although the
proportion of sterile granulomas is greater in those with LTBI
(39). Thus, unlike most infections, the clinical outcome of
infection is more accurately represented by the cumulative
host–pathogen interactions of all sites of involvement. That is,
all granulomas within a host need to either kill or control
M. tuberculosis bacilli to prevent development of disease; one
poorly functioning granuloma can lead to loss of containment
and TB progression.
The success or failure of each granuloma is dependent on the
immune response within that granuloma. The granuloma
functions to immunologically contain M. tuberculosis. Yet,
granulomas are heterogeneous in terms of cell types present
(T cells, B cells, macrophages, neutrophils), cellular immune
responses, and bacterial burden (47), and this heterogeneity
likely contributes to the variable range of infection outcomes
(Fig. 4). Surprisingly, ,10% of T cells within the granuloma
produce Th1 or Th17 cytokines (47). Our recent data suggest
that this is not due to exhaustion of T cells (49), as might be
expected in a chronic infection. Interestingly, the combina-
tion of IL-10–, TNF-, or IL-17–producing T cells, rather
than a preponderance of Th1 cytokines, within individual
granulomas was best associated with low bacterial burden and
sterile granulomas (47). Thus, the “success” (bacterial killing
or prevention of dissemination) of an individual granuloma
appears to be the result of both pro- and anti-inflammatory
processes rather than being depending on any single cytokine,
chemokine, or cell type. Although the host–pathogen inter-
action within an individual granuloma is relatively complex,
even greater complexity is observed at the organismal level
because of the number of heterogeneous granulomas present
within a single host.
This concept is important when examining the impact of
systemic immunologic interventions on individual granulomas
and on the overall outcome of the host. For example, the
granulomas during early infection are more homogeneous with
high bacterial burden and later become much more hetero-
geneous and likely less susceptible to immune suppression. For
example, depletion of CD4+ T cells during early M. tuberculosis
2545
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FIGURE 3. Granulomas are independent and dynamic by PET/CT during
M. tuberculosis infection in macaques. Top row; between 6 and 16 wk
postinfection, one granuloma increases in size but maintains the same FDG
avidity (yellow arrow), whereas the other granuloma increases in size and
FDG avidity (orange arrow) in the accessory lobe. Bottom row; this same
animal has a granuloma in the right lower lobe that has decreased in size and
FDG avidity (blue arrow) at the same timepoints. Black line = 1 cm.
infection resulted in 80% of animals with worsening TB pa-
thology, greater bacterial burden, and dissemination compared
with controls (50). In contrast, CD4+ T cell depletion during
LTBI resulted in only 50% of animals reactivating (50). Similar
findings have been observed with TNF neutralization, which
consistently exacerbates acute infection, but only results in
reactivation in ∼50% of LTBI macaques (4, 51). The greater
susceptibility to disseminated disease during early infection is
likely due to higher bacterial loads that require more of a robust
immune response to control within any individual granuloma
and the very low frequency of sterile granulomas at early
timepoints. For example, depletion of CD20+ B cells during
early infection did not result in overt clinical deterioration but
did significantly increase variability of bacterial burden and
influenced T cell responses within individual granulomas (52).
Thus, B cells may be important in stabilizing the granuloma
immune responses early in infection.
Outcomes in individual granulomas are likely associated with the TB
spectrum of risk. Given the heterogeneity of lesion types
seen among the various outcomes of M. tuberculosis infec-
tion, it is not surprising that the PET/CT patterns during
treatment would be extremely variable. Malherbe et al. (53)
characterized PET/CT patterns of active TB cases treated with
the standard four-drug regimen and correlated the findings
with clinical and microbiological outcomes. PET/CTs were
done at baseline, 1 and 6 mo after the start of treatment.
Six months after treatment, PET/CT patterns were classified
as resolved (i.e., minimal to no increase in FDG from
pretreatment baseline, regardless of CT abnormalities),
improved (i.e., decreased FDG intensity of all lesions but
with $1 lesion with increased FDG uptake), and mixed
(i.e., $1 lesion with higher FDG intensity compared with
baseline). Heterogeneous PET/CT scans were observed
2546 BRIEF REVIEWS: SPECTRUM OF M. TUBERCULOSIS INFECTION
FIGURE 4. The hypothetical spec-
trum of granuloma types is seen in any
infected individual with each gran-
uloma associated with a range of
immune control and bacterial burden.
A granuloma that has lost its overall
structure and function (progressive and
disseminating granulomas) is associated
with poor immune control and high
bacterial burden. In contrast, fibrotic
granulomas are often sterile reflecting a
robust immune response.
during treatment. Patients who failed treatment had
exclusively mixed patterns; however, mixed patterns could
also be seen among cured and recurrent patients. Moreover,
M. tuberculosis mRNA was identified in sputum 6 mo after
initiation of drug treatment in cured, failed, and recurrent
TB cohorts. The significance of this is yet unclear. The
spectrum of patterns seen during treatment likely reflects
the heterogeneous effect of drug treatment on individual
granulomas due to variable drug penetration into various
granuloma types (54, 55) and the efficacy of the individual
drugs against bacilli in different metabolic states depending
on the microenvironment of each granuloma (56) [reviewed
in (57)]. Clearly, more comprehensive studies are required to
better understand outcome and the appropriate treatment of
disease.
Reactivation risk in LTBI may well depend on a single
granuloma that is slightly impaired for bacterial control. In
LTBI, it traditionally was assumed that all granulomas are
successfully controlling or eliminating the infection. However,
the new paradigm states that LTBI exists as a spectrum of
infection outcomes. What defines the spectrum immunolog-
ically is not clear, but it is unlikely that this will be fully
understood simply from blood signatures. Data from our
macaque models indicate that immune responses in blood do
not reliably reflect immune responses in either individual
granulomas or the average of all granulomas (47). We per-
formed a large study of reactivation risk in LTBI cynomolgus
macaques, in which TNF was neutralized (a major risk factor
for reactivation TB in humans). PET/CT scans were per-
formed on macaques with LTBI over the 8-wk course of TNF
neutralization (4). Fifty percent of the macaques experienced
reactivated TB during this time. We assessed PET/CT fea-
tures prior to TNF neutralization that were associated with
reactivation and found that the number of granulomas in the
lung was not a factor. Instead, lung inflammation (often from
Downloaded from http://www.jimmunol.org/ by guest on October 22, 2018
a single granuloma) and the presence of an extrapulmonary
lesion (spleen or liver) predicted reactivation with 92% sen-
sitivity and specificity. To determine the importance of these
metrics, we predicted reactivation risk in a large number of
LTBI macaques who did not undergo TNF neutralization and
characterized their granulomas by PET/CT. The macaques
predicted to be at high risk using our PET/CT metrics had a
single granuloma with higher bacterial burden than those
predicted to be at low risk, supporting the concept that a
single granuloma can put a host at higher risk of reactivation
and that successful immune responses in all granulomas are
essential for long-term prevention of disease. However, the
immunologic microenvironment within granulomas is com-
plex and each granuloma likely takes its own path to success
or failure, making it unlikely that a single set of immune
responses uniformly leads to success (35). This paradigm has
major implications for vaccines and host-directed therapies,
as protective responses likely involve a variety of immune
mechanisms from different cell types.
Conclusions
Given the heterogeneous nature of bacterial dynamics and host
immune responses in individual granulomas, it is no wonder
that outcomes cannot be classified by binary outcomes of active
or LTBI. Moreover, these factors significantly complicate ef-
forts to improve TB treatment and develop protective vaccines.
Perhaps it will be more accurate to classify infection outcomes
as an individual’s risk of primary disease or reactivation. This
requires more precise diagnostics with greater specificity and
sensitivity, especially for immune-compromised hosts, such as
those with HIV who have the greatest risk of disease. It is
possible that risk assessments may require a series of tests
and rely on a battery of immune responses that could be
difficult to implement in the field instead of simply relying on
TST or IGRA results. Non–culture-based assays to identify
The Journal of Immunology
M. tuberculosis components, such as the urinary lipo-
arabinomannan assay (58) or trehalose assay (59), show
promise as a point-of-care diagnostic. Host-specific risks,
such as immune competence, age, genetic background, and
comorbidities, will need to be accounted for, not just once,
but over time as the risk can change over the life of the host.
That said, even with improved diagnostic methods and a
more thorough understanding of the disease and risk, we need
to ask ourselves some key questions. Given the complex
spectrum of M. tuberculosis infection, what will the optimal
treatment regimen look like? For example, what is the optimal
treatment for incipient disease? Is a four-drug therapy neces-
sary for a stage that may have a lower bacterial burden
than symptomatically overt, active TB? Will individualized
host-directed therapies, in conjunction with antimicrobial
treatment, shorten treatment duration? Because the disease
progression can change over time and the time of infection is
unknown, do patients require repeated testing for incipient
and overtly active TB? Will there be an accurate and cost-
effective method for identifying those at low-to-no risk of
reactivation? Does that risk change over time? Can tests of
incipient TB that all currently rely on transcriptional signa-
tures be converted to point-of-care assays that are practical in
resource-limited settings? Clearly more research is required to
better understand the pathogenesis and immune responses
that categorize status of infection and risk of disease on both a
microscopic (e.g., within the granuloma) and macroscopic
(clinical outcomes) level to drive better diagnostic and ther-
apeutic modalities for the future.
Acknowledgments
We thank all the members of the Flynn and Lin laboratories and our colleagues
Drs. Sarah Fortune, Denise Kirschner, Charles Scanga, Edwin Klein, Hannah
Gideon, and Joshua Mattila for helpful discussions that contributed to this
review. Special thanks to Elise Y. Chu and Pauline Maiello for artistic and
graphic contributions.
Disclosures
The authors have no financial conflicts of interest.
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