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Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2571-2580
Host Reactive Donor T Cells Are Associated With Lung Injury After
Experimental Allogeneic Bone Marrow Transplantation
By
Kenneth R. Cooke,
Werner Krenger,
Geoff Hill,
Thomas R. Martin,
Lester Kobzik,
Joanne Brewer,
Raymond Simmons,
James M. Crawford,
Marcel R.M. van den Brink, and
James L.M. Ferrara
From the Department of Pediatric Oncology, Dana-Farber Cancer
Institute; the Department of Pathology, Brigham and Women's Hospital;
the Division of Pediatric Pulmonology, Children's Hospital, Boston,
MA; and the Department of Pathology, Yale University School of
Medicine, New Haven, CT.
 |
ABSTRACT |
Noninfectious lung injury is common after allogeneic bone marrow
transplantation (BMT), but its association with acute graft-versus-host disease (GVHD) is unclear. Using a murine BMT system where donor and
host differ by multiple minor histocompatibility (H) antigens, we
investigated the nature of lung injury and its relationship both to
systemic GVHD and host-reactive donor T cells. Lethally irradiated
CBA hosts received syngeneic BMT or allogeneic (B10.BR) T-cell-depleted (TCD) bone marrow (BM) with and without the addition of T cells. Six weeks after BMT, significant pulmonary histopathology was observed in animals receiving allogeneic BMT compared with syngeneic controls. Lung damage was greater in mice that received allogeneic T cells and developed GVHD, but it was also detectable after
TCD BMT when signs of clinical and histologic acute GVHD were absent.
In each setting, lung injury was associated with significant
alterations in pulmonary function. Mature, donor (V 6+
and V3+) T cells were significantly increased in the
broncho-alveolar lavage (BAL) fluid of all allogeneic BMT
recipients compared with syngeneic controls, and these cells
proliferated and produced interferon- (IFN-) to host antigens in
vitro. These in vitro responses correlated with increased IFN- and
tumor necrosis factor- (TNF-) in the BAL fluid. We conclude that
alloreactive donor lymphocytes are associated with lung injury in this
allogeneic BMT model. The expansion of these cells in the BAL fluid and
their ability to respond to host antigens even when systemic tolerance has been established (ie, the absence of clinical GVHD) suggest that
the lung may serve as a sanctuary site for these host reactive donor T
cells. These findings may have important implications with regard to
the evaluation and treatment of pulmonary dysfunction after allogeneic
BMT even when clinical GVHD is absent.
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INTRODUCTION |
LUNG INJURY IS a frequent
and severe complication following allogeneic bone marrow
transplantation (BMT). Pulmonary insults in various forms occur in 25%
to 55% of transplanted patients and account for approximately 40% of
transplant-related mortality.1-6 Idiopathic pneumonia
syndrome (IPS) refers to diffuse noninfectious pneumonia that occurs in
this setting.1 A recent study showed a lower incidence and
earlier onset of IPS than previously reported, but the typical clinical
course involving the rapid onset of respiratory failure leading to
death remained unchanged, underscoring the critical nature of this
transplant-related problem.6 Although IPS has been
associated with the development of clinical and experimental acute
graft-versus-host disease (GVHD),1,5-8 it has also been reported after allogeneic T-cell-depleted (TCD) BMT when signs and
symptoms of GVHD are absent,9,10 making a causal
relationship between the two entities difficult to establish. We have
developed a murine model of IPS where GVHD and lung injury are induced
by minor histocompatibility (H) antigenic differences between donor and
host (B10.BR  CBA) and have previously described
the inflammatory aspects of the pulmonary damage.7,11 Using
this BMT system, we have now examined the relationship of lung injury
to alloreactive donor T cells and to acute GVHD. Our data show that
noninfectious lung injury that occurs after allogeneic BMT is
associated with an expansion of host-reactive donor T cells in the
broncho-alveolar lavage (BAL) fluid and results in significant
alterations in pulmonary function. Depletion of donor T cells in the
bone marrow inoculum at the time of transplant significantly reduces
but does not eliminate this injury, even though it prevents the
development of acute GVHD. Host-reactive donor T cells were
identified in the BAL fluid but not the spleens of TCD BMT recipients,
which helps to explain the dichotomous nature of injury to host tissues
and which suggests that the lung may be a particularly reactive
site for donor T lymphocytes after allogeneic BMT.
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MATERIALS AND METHODS |
Mice, BMT, and assessment of GVHD.
The protocol for BMT and GVHD induction has been described
previously.12,13 Female CBA/J (H-2k) and B10.BR
(H-2k) mice were purchased from the Jackson Laboratories
(Bar Harbor, ME) and were transplanted between the ages of 10 and 20 weeks. Bone marrow (BM) was obtained from the femurs and tibias of
donor CBA or B10.BR mice and depleted of T cells using an anti-Thy 1.2 monoclonal antibody (MoAb; American Type Culture Collection, Rockville, MD) and Low-Tox-M rabbit complement C (Accurate Corp, Westbury, NY).
We have previously shown that TCD with two rounds of anti-Thy 1.2 MoAb
and complement leaves fewer than 1 in 104 mitogen
responsive T cells in the BM by limiting dilution assay.14 Cell mixtures of 5 × 106 BM cells supplemented with 1 × 106 nylon wool nonadherent splenic T cells from
either syngeneic (CBA) or allogeneic (B10.BR) donors were resuspended
in Leibovitz's L-15 medium (Life Technologies, Grand Island, NY) and
transplanted into CBA recipients via tail vein infusion (0.25 mL total
volume). Other CBA animals received allogeneic TCD BM only. Before
transplant, host mice received 11 Gy of total body irradiation
(137Cs source) delivered in two fractions, separated by 3 hours to reduce gastrointestinal toxicity. This dose of irradiation
does not cause histologically detectable pulmonary injury in normal CBA
mice.14 Mice were subsequently housed in sterilized
microisolator cages and received normal chow and autoclaved
hyperchlorinated water for the first 2 weeks post-BMT and filtered
water thereafter.
The severity of GVHD was assessed by percent weight change (recipient
mice were ear punched and individual weights were obtained and recorded
on day +1 and weekly thereafter until the time of analysis) and a
clinical scoring system previously described that incorporates five
clinical parameters: weight loss, posture (hunching), activity, fur
texture, and skin integrity.7 At the time of analysis, mice
from coded cages were evaluated and graded from 0 to 2 for each
criterion. A clinical index was subsequently generated by summation of
the five criteria scores (maximum index = 10).
Tissue procurement and histopathologic analysis of GVHD.
The presence of acute GVHD was also assessed by detailed
histopathologic analysis of classic target organs. Samples of oral mucosa and a portion of the right lobe of the liver were obtained from
animals 6 weeks after BMT and placed in buffered formalin. Formalin-preserved specimens were then embedded in paraffin, cut into 5 µm thick sections, and stained with hematoxylin and eosin for
histological examination. Slides were coded without reference to mouse
type or prior treatment status and systematically examined by
pathologists (oral mucosa, R.S.; liver, J.M.C.) to establish an index
of injury. Samples of oral mucosa were analyzed by counting the total
number of lymphocytes present per high-powered field as previously
described, and averages were taken from four to five fields per
section.13 Analysis of liver tissue was performed by
scoring 14 pathologic features, including inflammatory infiltrates in
bile ducts and portal tracts, vascular endothelialitis, and hepatocellular damage as previously reported.11,13 A
severity scale from one to four was used where 0 = normal, 0.5 = rare
scattered, 1= minimal or focal, 2 = mild and more diffuse, 3 = moderate
damage, and 4 = severe damage. Scores for each individual feature were added to yield a composite score of liver pathology.
Examination of lung histopathology and measurement of pulmonary
function.
The presence of pulmonary toxicity after BMT was determined by
examination of lung histopathology and pulmonary function in transplanted animals 6 weeks after BMT. Lungs from each mouse were
inflated with 1 mL of Tissue Tek OCT compound (Miles, Elkhart, IN) and
removed from the thoracic cavity. The right lower lobe and left lung
were immersed in 10% buffered formalin. Formalin-preserved specimens
were then embedded in paraffin, cut into 5 µm thick sections, and
stained with hematoxylin and eosin for histological examination. Slides
were coded without reference to mouse type or prior treatment status
and systematically examined by L.K. to establish an index of injury.
Lung tissue was evaluated for the presence of periluminal infiltrates
(around airways and vessels) or parenchymal pneumonitis (involving the
alveoli or interstitium) using a semiquantitative scoring system as
previously described that incorporates both the severity and extent of
histopathology.7
Dynamic pulmonary compliance (Cdyn) and airway conductance
(GL) were measured in live mice using a
plethysmographic technique as previously described.15,16 A
state of general anesthesia was induced using 70 to 90 mg/kg of
pentobarbital injected intraperitoneally, and 1% lidocaine was used as
an additional local anesthetic in the region of the anterior neck. A
19-gauge tubing adapter (used as a tracheostomy tube) was then
surgically inserted into the trachea and secured with silk suture.
Mechanical ventilation was instituted with a rodent ventilator (Harvard
Apparatus, Natick, MA) set to deliver a tidal volume of 0.07 mL per 10 g of body mass at a frequency of 150 breaths per minute and a positive
end-expiratory pressure of 2 to 3 cm H20. A small portion
of the thoracic wall was excised to eliminate any effect of the chest
wall on pulmonary function. The mice were placed in a
plexiglas chamber, which was subsequently sealed to a
constant volume plethysmograph system. Pressures in the chamber and in
the tracheostomy tube were detected by separate transducers (Celesco,
Canoga Park, CA), amplified, and converted from analog to digital data
to be processed by a computer programmed to calculate Cdyn
and GL.15,16 Thirty seconds before each
determination, a deep inspiration (3 to 4 × tidal volume) was
delivered to provide a constant volume history and to prevent
atelectasis. Measurements of Cdyn and GL were
obtained at 1-minute intervals for 5 minutes for each mouse and the
results averaged. The coefficient of variation for values of
Cdyn and GL from individual mice was less than
5%. Standard curves for both measures of pulmonary function were
generated for CBA mice by measuring Cdyn and GL
in naive animals from age 10 to 32 weeks at 2- to 4-week intervals (n = 4 to 5 animals per age group). Measurements of Cdyn and
GL obtained from transplanted animals were therefore
compared with mean values for age-matched naive controls, and the
results are expressed as the percent predicted score.
Broncho-alveolar lavage (BAL), cytokine determination, and cellular
differential.
After the determination of pulmonary function, mice were killed by
exsanguination and BAL was performed. A 0.8 mL aliquot of 1×
phosphate buffered saline (PBS) containing 0.6 mmol/L EDTA was
instilled into the lungs through the secured tracheostomy tube, of
which 0.7 mL was removed and placed into a sterile tube on ice. This
procedure was repeated nine additional times with subsequent aliquots
combined in a second tube. The tubes were centrifuged at 1,500 rpm for
5 minutes, and supernatant from the first tube was frozen for
subsequent analysis. Cell pellets from both tubes were combined, washed
twice, and counted. Aliquots of cell suspensions (2 × 106 cells per mL) were placed on glass cover slips,
air-dried, stained with Wright-Giemsa, and mounted on microscope
slides. Coded slides were then evaluated visually for morphologic
differentials. Concentrations of tumor necrosis factor- (TNF-)
and interferon- (IFN-) were measured in BAL fluid supernatant
(obtained from the first laved aliquot) by sandwich enzyme-linked
immunosorbent assay (ELISA) using specific anti-murine MoAbs for
capture and detection and the appropriate standards purchased from
Pharmingen (IFN-; San Diego, CA) and Genzyme (TNF-; Cambridge,
MA). Assays were performed according to the manufacturer's protocol.
Lower limits of detection of these assays were 15 pg/mL (TNF-) and
0.25 U/mL (IFN-). For determination of endotoxin concentration in
BAL fluid, the limulus amebocyte lysate (LAL) assay QLC-1000 test kit
was used (Bio Whittaker, Walkersville, MD). Assays were performed
according to the manufacturer's protocol as previously
described.7 In all cytokine and endotoxin determinations,
samples, and standards were run in duplicate.
Cell culture, analysis of proliferative response, and IFN-
production.
All culture media reagents were purchased from GIBCO-BRL (Gaithersburg,
MD). For analysis of proliferative response and IFN- production, BAL
cells and splenocytes were obtained from individual recipients 6 weeks
post-BMT and pooled within treatment groups. Splenocytes (ranging in
concentration from 0.5 to 2.0 × 105) were cultured in
flat-bottomed 96-well Falcon plates (Lincoln Park, NJ) for 72 hours in
the presence of irradiated and TCD (anti-Thy 1.2 MoAb and
C) stimulator cells from CBA/J mice (2 × 105/well). Cell culture was performed in 5% fetal calf
serum (FCS)/RPMI supplemented with 50 U/mL penicillin, 50 µg/mL
streptomycin, 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, 0.1 mmol/L nonessential amino acid, 0.02 mmol/L -mercaptoethanol, and 10 mmol/L HEPES, pH 7.75 at 37°C in a humidified incubator
supplemented with 7% CO2. BAL cells were preincubated for
90 minutes at 37°C to remove adherent macrophages and then cultured
in a similar manner. Supernatants were collected at 48 hours for
IFN- analysis by ELISA, and proliferative response to host antigen
was measured after 72 hours by incorporation of [3H]
thymidine (1µCi) for the last 24 hours of incubation.
Statistical considerations.
All values are expressed as the mean ± SEM. Statistical comparisons
between groups were completed using the nonparametric, unpaired,
Mann-Whitney test.
| |
RESULTS |
Significant GVHD and lung injury develops after allogeneic BMT.
CBA mice were first transplanted with syngeneic (CBA) or allogeneic
(B10.BR) TCD bone marrow and 1 × 106 donor T cells.
Animals were assessed weekly for systemic GVHD using percent weight
change and the clinical scoring system described in Materials and
Methods. Recipients of allogeneic BM and T cells developed significant
weight loss within the first week after BMT and, despite a transient
recovery, continued to lose weight throughout the observation period
consistent with the development of acute GVHD
(Fig 1A, P < .01). Approximately
40% of these animals died of GVHD by 6 weeks after BMT (data not
shown). By contrast, mice receiving syngeneic BMT all survived and were
at or above their pretransplant weight 6 weeks after BMT. Recipients of
allogeneic BMT also developed significant signs of GVHD compared with
syngeneic controls as assessed by alterations in fur texture, skin
integrity, posture, mobility, and weight (Fig 1B, P < .01).
The extent of acute GVHD was further evaluated 6 weeks after BMT by
histopathologic evaluation of target organ tissue as described in
Materials and Methods. As shown in Fig 1C and D, semiquantitative
analysis showed significant tissue injury in the liver and oral mucosa
of animals receiving allogeneic BM and 1 × 106 donor
T cells compared with recipients of syngeneic BMT (P < .01),
consistent with the presence of clinical GVHD in these animals.
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| Fig 1.
Analysis of systemic (A and B) and target organ (C and D)
GVHD after BMT. CBA mice received syngeneic TCD BM and T cells  , TCD
allogeneic (B10.BR) BM alone  , or with the addition of 1 × 106 T cells  , as described in Materials and Methods.
Recipients of allogeneic BM and T cells developed significant GVHD as
determined by weight loss (A) and clinical score (B), compared with
recipients of syngeneic BMT (**P < .01). The extent of acute
GVHD was also assessed 6 weeks after BMT by histopathologic evaluation
of target organ tissue as described in Materials and Methods. Six weeks
after transplant, recipients of allogeneic BM and T cells developed
significant tissue injury in the liver (C) and oral mucosa (D) compared
with syngeneic BMT controls (**P < .01). Transplantation of
allogeneic TCD BM alone completely prevented the development of GVHD;
weights, clinical scores, and target organ pathology scores of these
animals were indistinguishable from syngeneic controls. Data are
expressed as mean ± SEM. (A and B, n = 12 to 15 per group; C and D,
n = 6 to 9 per group)
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To evaluate the extent of pulmonary toxicity that developed 6 weeks
after BMT, lung tissue was obtained, examined microscopically, and
scored semiquantitatively as described in Materials and Methods. Consistent with previous studies, lungs of mice receiving syngeneic transplants maintained virtually normal histology, whereas two major
abnormalities were apparent in the group receiving allogeneic BM and T
cells.7 First, a dense mononuclear cell infiltrate was
found around both pulmonary vessels and bronchioles, and second, an
acute pneumonitis was observed involving both the interstitium and
alveolar spaces. The alveolar infiltrate was composed of macrophages, lymphocytes, epithelial cells, and scattered polymorphonuclear cells
within a fibrin matrix (data not shown). Semiquantitative evaluation of
lung sections showed that significant pulmonary damage was present
after allogeneic BMT compared with syngeneic controls
(Fig 2A, P < .01).
Potential infectious causes of pulmonary injury were ruled out by
screening sentinel mice from each transplant group for a panel of both
viral and bacterial organisms as previously described.7 No
pathogenic organisms were identified in any mice, and lung tissue was
negative for Pneumocystis carinii by silver staining.
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| Fig 2.
Semiquantitative analysis of lung
histopathology (A) and pulmonary function (B and C) 6 weeks after BMT.
CBA mice received BMT as in Fig 1 (syngeneic , allo TCD BM alone
, allo TCD BM and T cells ). Lungs were obtained after BMT and
analyzed as described in Materials and Methods. Significant lung injury
was present in animals receiving allogeneic T cells compared with
syngeneic BMT recipients (**P < .01). TCD significantly
reduced (#P < .01), but did not eliminate, lung injury and
pathology scores after TCD BMT remained significantly increased
compared with syngeneic controls (*P < .01). Pulmonary
dynamic compliance (Cdyn) and airway conductance
(GL) were measured in live transplanted animals as
described in Materials and Methods. Lung injury that developed in mice
with GVHD was associated with significant decreases in Cdyn
and GL compared with syngeneic controls (**P < .01). Mice receiving TCD BMT showed significant improvements in
Cdyn (B) but not GL (C) compared with animals
with GVHD (#P = .01), but both measurements remained
significantly reduced compared with syngeneic controls (*P < .01). Data are expressed as mean ± SEM. (n = 12 to 15 per group).
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We next evaluated the physiologic consequences of the lung pathology
present after BMT by measuring pulmonary dynamic compliance (Cdyn) and airway conductance (GL) in live
transplanted mice using the well-established plethysmographic technique
described in Materials and Methods. Abnormalities in Cdyn,
defined as the change in lung volume resulting from a given increase in
distending transpulmonary pressure, would be expected with pulmonary
parenchymal alterations, including consolidation, atelectasis, and
interstitial inflammation or fibrosis. GL, measured as flow
per unit pressure and which represents the reciprocal of resistance,
would be influenced by conditions that result in alteration of
bronchial diameter. Six weeks after transplant, mice receiving
allogeneic BMT showed significant reductions in both dynamic compliance
and airway conductance compared with syngeneic controls consistent with
both the interstitial and peribronchial infiltrates seen
microscopically (Fig 2B and C, P < .01).
TCD prevents GVHD and significantly reduces, but does not eliminate,
lung injury after BMT.
We analyzed the contribution of donor T cells to GVHD and lung injury
that developed after allogeneic BMT by using the method of TCD
described in Materials and Methods. CBA recipients of allogeneic TCD BM
alone (TCD BMT) at no time showed evidence of active GVHD, and their
weight curves and clinical scores at the time of analysis were
indistinguishable from syngeneic controls (Fig 1A and B). Similarly,
target organs from recipients of TCD BMT had equivalent pathology
scores compared with syngeneic controls (Fig 1C and D), confirming the
clinical analysis and consistent with multiple studies that TCD BMT
prevents GVHD.13,17,18 As shown in Fig 2A, evaluation of
lung sections showed that TCD significantly reduced, but did not
eliminate, the development of lung injury after BMT (P
<0.01). This damage was manifest primarily as mononuclear cell
infiltration around pulmonary vessels and bronchi, while a mild
pneumonitis was seen in the alveoli and interstitium of approximately
50% of animals (data not shown). As shown in Fig 2B and C, analysis of
pulmonary function confirmed these surprising results; recipients of
TCD BMT showed significant improvements in Cdyn (P
= .01) but not in GL compared with mice with GVHD, and
both of these measurements remained significantly reduced compared with
syngeneic controls (P < .01). These pulmonary function test
findings correlated well with the extent and nature of histologic lung
damage present after TCD BMT and confirmed that these animals develop
significant lung toxicity in the absence of systemic and histopathologic GVHD as measured in usual target organs (Fig 1C and D).
Lung injury in recipients of allogeneic BMT is associated with
increases of TNF-, IFN-, and host-reactive T cells in the BAL
fluid.
In an attempt to delineate the mechanisms responsible for lung injury
occurring after allogeneic BMT, BAL fluid was collected from all
transplanted mice before fixation of lung tissue as described in
Materials and Methods. We have previously shown that inflammatory changes in BAL fluid correlate with this injury,7 and
therefore we evaluated cellularity, cytokine content, and LPS levels in the BAL fluid of all transplanted mice. As shown in
Table 1, lung injury in recipients of
allogeneic BM and T cells that developed GVHD was associated with a
significant increase in the number of total BAL cells compared with
syngeneic controls (P < .01). Also noted in this group was a
fivefold increase in lymphocytes, and significant increases in
macrophages and neutrophils, consistent with the mixed inflammatory
alveolar infiltrates observed on histopathology. BAL fluid cellularity
remained significantly elevated in recipients of TCD BMT compared with
syngenic controls (P < .01) and included increased numbers of
both neutrophils and lymphocytes (P < .05). Nearly identical
changes were noted with respect to BAL TNF-, which was reduced after
TCD BMT by more than 50% (Fig 3A,
P = .01) but which remained significantly elevated compared
with syngeneic controls (Fig 3A, P < .05). Despite the
increased levels of both neutrophils and TNF-, LPS levels in the BAL
fluid from mice receiving TCD BMT were not elevated compared with
syngeneic controls (data not shown). We hypothesized that the increase
in TNF- in the absence of LPS in BAL fluid after TCD BMT might be
derived from T cells or activated "primed" pulmonary macrophages.
We therefore analyzed BAL fluid IFN- levels to evaluate this
hypothesis. As shown in Fig 3B, IFN- levels in the TCD BMT group
were significantly increased compared with syngeneic recipients
(P < .01), and somewhat surprisingly, were comparable with
those measured in animals with GVHD. We next investigated whether T
cells in BAL fluid might represent a source of this IFN-. Responses
of BAL lymphocytes to alloantigens in vitro have been used as a
reliable indicator of both acute and chronic rejection after lung
transplantation,19 and we therefore evaluated the responses
of BAL lymphocytes to host antigens in this BMT model. BAL cells
obtained from transplant recipients at week 6 were cultured in vitro
with host stimulator cells, and as shown in
Fig 4A, BAL cells from mice with GVHD
responded vigorously. The proliferative response of these cells was
sixfold higher than syngeneic controls, and their production of IFN- was markedly increased (20.3 ± 1.2 v ND). TCD reduced but
did not eliminate these responses, as proliferation and IFN-
production to host antigens by BAL cells from recipients of TCD BMT
remained significantly increased compared with syngeneic controls.
Thus, host-reactive donor T cells appeared to contribute to the BAL fluid changes and pulmonary damage observed after allogeneic BMT.
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| Fig 3.
BAL fluid TNF- and IFN- concentrations after BMT.
CBA mice received BMT as in Fig 1 (syngeneic , allo TCD BM alone
, allo TCD BM and T cells ). BAL fluid was obtained 6 weeks after
BMT and analyzed for TNF- and IFN- concentrations as described in
Materials and Methods. Both TNF- and IFN- were significantly
increased in the BAL fluid from mice receiving allogeneic BM and T
compared with syngeneic controls (**P < .01). BMT with TCD BM
only lead to a 50% reduction in TNF- (#P = .01) but not
IFN-, and levels of both cytokines remained significantly elevated
compared with syngeneic controls (*P < .05). Data are
expressed as mean ± SEM. (n = 8 to 12 per group).
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| Fig 4.
Proliferation and IFN- production of BAL fluid (A) and
Splenic (B) T cells to host (CBA) antigens in vitro. CBA mice received
BMT as in Fig 1 (syngeneic, allo TCD BM alone  , allo TCD BM and
T cells ). BAL cells and splenocytes were obtained from transplanted
animals 6 weeks after BMT and cultured with irradiated host stimulator
cells as described in Materials and Methods. BAL and splenic T-cell
populations from recipients of allogeneic BM and T cells responded to
host antigens in vitro. TCD reduced, but did not eliminate,
proliferation and IFN- production of pulmonary T cells to host
antigens, whereas similar responses by splenocytes were absent,
consistent with the lack of GVHD in these animals. Values for
proliferation were normalized for the percent T cells present in each
group (CD4+ and CD8+). IFN- levels
expressed in U/mL were measured at maximum proliferative response (200 × 103 responders per well). Data are expressed as mean ± SEM of triplicate wells and represent one of two similar
experiments.
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The response to host antigens by BAL fluid T cells after TCD BMT was
somewhat surprising because very few T cells remained in the BM
inoculum after the TCD procedure, and the effectiveness of that
protocol was confirmed by the absence of clinical or histologic GVHD in
these animals (Fig 1). To determine whether T-cell reactivity to host
antigens was confined to the lungs of these animals, similar in vitro
cultures were performed using splenocytes from transplanted animals as
responders to host antigens in a mixed lymphocyte reaction (MLR). As shown in Fig 4B, the proliferative response of
splenocytes from TCD BMT recipients to host antigens was equivalent to
that of syngeneic controls. IFN- production was also undetectable in
supernatants of these splenocyte cultures, consistent with the absence
of systemic GVHD. By contrast, splenocytes from animals with GVHD both
proliferated and produced IFN- when stimulated with host antigens.
Interestingly, BAL cells from mice with GVHD produced approximately
sevenfold more IFN- than did splenocytes from the same animals.
Taken together, these findings show that T cells that are responsive to
host antigens are present in the lung after allogeneic BMT and that
T-cell alloreactivity may be enhanced there even when such responses
are low or undetectable in the periphery.
Donor V6+/TCR+ T cells expand in the
lungs after allogeneic BMT.
The above responses to host antigens in vitro strongly suggested the
presence of donor T cells in the BAL fluid of mice after allogeneic
BMT. To confirm the origin of these cells, we used differences in the T
cell V repertoire between donor and host. Mature V6+
and V3+ T cells are normally deleted by negative
selection in the thymuses of host (CBA) but not donor (B10.BR) animals
due to differences in Mls expression.20 However, both
V6+ and V3+ T-cell populations, which
recognize CBA antigens in vitro, have been found in the peripheral
circulation of CBA recipients of allogeneic BMT as a result of thymic
dysfunction and damage associated with the development of
GVHD.21 We therefore analyzed BAL fluid and splenic T-cell
populations by flow cytometry for the expression of V6, V3, and
TCR antigens. As shown in Table 2,
the percentage of splenic V6+ T lymphocytes was
significantly increased in mice with GVHD compared with both syngeneic
and TCD BMT recipients (P < .01). Dramatic increases in the
percentage of donor T cells were also seen in the BAL fluid of these
mice; nearly 40% of TCR+ T cells were
V6+, a level fourfold higher than in naive B10.BR mice.
Consistent with their reactivity to host antigens in vitro,
approximately 21% of BAL fluid TCR+ lymphocytes from
TCD BMT recipients also expressed V6, confirming the donor origin of
those T cells (P < 0.01, compared to syn). A smaller albeit
significant increase in V6+ cells was apparent in the
splenic T-cell population of these animals (5.5 ± 0.8% v
0.7 ± 0.1%), where donor tolerance of host antigens was
demonstrable in vitro (Fig 4B) and confirmed in vivo by the absence of
systemic or histologic GVHD (Fig 1A through D). Thus,
V6+ donor T cells homed to the lungs after allogeneic
BMT, persistently responded to host antigens, and were associated with
clinically and histologically significant tissue injury. Depletion of
donor T cells at the time of BMT reduced, but did not abrogate, donor T-cells response in the lungs even though the number of T cells in the
donor BM inoculum was insufficient to cause clinical or histologic GVHD
and the small percentage of donor T cells identified in the spleens of
these animals did not recognize host antigens in vitro.
| |
DISCUSSION |
Using a well-characterized murine BMT model, we have examined the
nature of lung histopathology that develops after allogeneic BMT and
its relationship to both acute GVHD and host-reactive donor T cells.
Our data show that significant noninfectious lung damage occurs in
animals with GVHD (Fig 2). This injury is associated with an expansion
of host-reactive donor (V6+) T cells (Fig 4, Table 2)
and increases in TNF- and IFN- in the BAL fluid of affected
animals (Fig 3). The physiologic significance of this injury is
underscored by abnormalities in pulmonary function, where both dynamic
compliance and airway conductance were decreased after BMT (Fig 2).
Depletion of donor T cells at the time of BMT significantly reduced but
did not eliminate this injury, even though it effectively prevented the
development of systemic GVHD (Figs 1 and 2). The residual lung injury
after TCD BMT resulted in alteration in pulmonary function,
particularly with respect to airway conductance, which failed to
improve compared with mice with GVHD (Fig 2). Interestingly, an
expansion of host-reactive donor V6+ T cells was
identified in the BAL fluid but not in the spleens of TCD BMT
recipients, consistent with the lack of clinical and histologic GVHD
seen in these animals. These results support the hypothesis that
host-reactive donor T cells can significantly contribute to
noninfectious lung injury that occurs after BMT. They also show that
the lung is susceptible to immune-mediated damage from small numbers of
donor T cells present following allogeneic TCD BMT and suggest the lung
may represent a sanctuary site for donor lymphocytes even when systemic
tolerance between donor and host is established.
The role of GVHD and specifically of alloreactive donor T cells in the
pathogenesis of IPS remains a topic of considerable debate. Although a
variety of pulmonary complications and histopathologic findings have
been associated with the development of GVHD, a mechanistic
relationship between noninfectious lung injury and immunologically
active donor T cells has not been clearly established. The principal
objection to the identification of the lung as a target organ of acute
GVHD is that epithelial cell apoptosis, a finding classically
attributed to selective T-cell-mediated injury and considered
pathognomonic for acute GVHD in the gut, liver, and skin of patients
after BMT, has not been consistently identified among the myriad of
histologic findings noted in the lungs of patients with
IPS.22-25 In 1978, Beschorner et al22 noted an
association between the severity of clinical GVHD and a histologic
pattern consistent with lymphocytic bronchitis found on postmortem
exams. This finding was not seen in patients who received autologous
BMT or in untransplanted controls.22 Although initially
considered a potential histopathologic correlate for GVHD of the lung,
the association between lymphocytic bronchitis and the development of
systemic GVHD was not consistently identified in subsequent
reports.23-25
However, the heterogeneity of pulmonary histopathology in clinical BMT
is complicated by both the nonspecific changes that occur after
mechanical ventilation and the suboptimal quality and quantity of
pathology specimens due to the significant risks associated with lung
biopsy procedures. Despite the lack of classic GVHD
histopathology, it is not unreasonable to suggest that pulmonary parenchymal and endothelial cells can be potential targets for activated donor T cells after allogeneic BMT. Firstly, the
lung is a rich source of major and minor HC antigens and professional antigen-presenting cells,26,27 and it is the site of
complex immunologic networks, the proper balance of which allows for
infectious surveillance and maintenance of structural integrity,
whereas dysregulation of such networks can result in tissue injury and scarring.28 The role of T lymphocytes to immune-mediated
inflammatory reactions in the lung has recently been confirmed by
several groups and is thought to involve dendritic cells, macrophages,
and the secretion of cytokines that can influence an emerging T-cell
response.29,30 Secondly, the association of chronic GVHD
with obstructive lung disease after BMT is well
accepted.31-36 Although a causal link between these two
entities has yet to be definitively established, the striking
similarities between the consistent histopathologic features of
obstructive bronchiolitis after BMT and the bronchiolitis obliterans
associated with lung transplant rejection along with reports of
improvement in lung function with immunosuppressive agents strongly
suggests an immunological component to this pulmonary process.31,35-36 It would be inconsistent for the lung to
be a target of a chronic but not an acute immune attack. Thirdly,
epithelial cell apoptosis is not a requirement of GVHD pathology. The
thymus is a known target of GVHD and displays extensive cytolytic
damage early in the course of this process, but epithelial cell
apoptosis is not a prominent histologic feature.37 Finally,
recent studies have shown that GVHD target organs vary with respect to
their susceptibility to injury by inflammatory effectors such as CTLs, TNF-, and FasL.38,39 If the mechanisms of GVHD-related
tissue injury can differ between individual target organs, it is
possible that the histopathologic manifestation of this injury may also vary.
Recently, several studies using established rodent BMT models to
explore the potential causal relationship between GVHD and IPS have
shown the development of pulmonary injury in animals with systemic
GVHD.7,40-42 Advantages of these systems include the
unlimited availability of tissue for pathologic analysis and the
ability to analyze the development of tissue injury without the
confounding influences of immunosuppressive chemoprophylaxis, underlying disease, or prior treatment. Surprisingly, even under controlled experimental conditions, several patterns of lung injury have been identified. For example, using a B10 (CBA × B10) F1 murine BMT model, Piguet and coworkers40 observed
both an acute hemorhagic alveolitis and a late onset interstitial
pneumonitis (IP) after infusion of B10 parental lymphocytes, whereas
induction of GVHD with T cells from CBA donors led to IP only.
Subsequently, the development of interstitial pneumonitis and
lymphocytic bronchiolitis/bronchitis similar to the histopathology seen
in lung allograft rejection was noted in an unirradiated rat GVHD
model.41 We have observed similar pulmonary pathology in
the murine BMT system studied here and have consistently identified
parenchymal pneumonitis and mononuclear cell infiltration around both
bronchial and vascular structures as the two major microscopic patterns
of injury.7
Experimental models have also provided insight into the possible
pathophysiologic mechanisms responsible for noninfectious lung injury
occurring after BMT. TNF- has been detected in the lungs of animals
with GVHD,40,43 and administration of TNF antiserum blocked
the development of alveolar hemorrhage but did not reduce the
accompanying cellular infiltrate.40 We have previously shown that lung injury that occurred during active GVHD was associated with increased BAL fluid levels of neutrophils, TNF-, and
LPS.7 Elevations of proinflammatory cytokines in the lungs
of patients with pneumonitis has been confirmed in one report
44 but not in another where lung toxicity after BMT was
associated with a Th-2 cytokine response.45 The importance
of lymphocytes to lung injury after BMT has also been suggested by
several groups.42,46-47 Panoskaitsis-Mortari and
coworkers42 showed that donor T cells were important
mediators of lung toxicity that developed within the first week of BMT
across MHC antigens. Additionally, donor T-cell clones that recognize
CD45 polymorphisms contributed to a rapidly progressive pulmonary
vasculitis within the first 3 days after their injection into
nonirradiated recipients.47 These findings support the
clinical observation of Leblond and colleagues,48 who
suggested that the alveolar lymphocytosis that accompanied interstitial
pneumonitis after allogeneic BMT could represent a pulmonary
manifestation of chronic GVHD.
The current study further dissects the pathogenesis of IPS by showing a
role for donor T-cell reactivity in mediating lung damage that occurs
after allogeneic BMT, even when systemic GVHD is absent. Our
observation that host-reactive donor lymphocytes are present in the BAL
fluid but not the spleens of animals after TCD BMT is intriguing and
suggests that the lung may be particularly sensitive to the effects of
these cells even when systemic tolerance has been established. It
remains unresolved, however, as to whether the GVHD induced alterations
in BAL fluid cytokine levels and in pulmonary histology after TCD BMT
are mediated by mature donor T cells remaining in the BM innoculum, by
effector cells that have differentiated from an engrafted BM within an
allogeneic thymic environment, or by a combination of these cells. With
respect to the latter, previous work using this strain combination has shown that acute GVHD is accompanied by loss of normal thymic repertoire selection.21 In the current study, thymic
cellularity was reduced in mice receiving TCD BMT compared with
syngeneic BMT controls (26.3 ± 2.5 × 106
v 42.9 ± 3.9 × 106, P < .01),
whereas the number of thymocytes in both groups remained significantly
higher than those in mice with clinically evident GVHD (6.4 ± 2.0 × 106, P < .01). Thus, mild thymic
dysfunction and a loss of negative selection could account for the
persistence of host-reactive donor T cells after TCD BMT. Data
supporting the second hypothesis can be found in an study by Korngold
et al,17 where as few as 3 × 104 B10.BR T
cells (equivalent to 0.3% T-cell contamination of the marrow) resulted
in significant systemic disease and mortality from GVHD in CBA
recipients. It is possible, therefore, that transplantation of a very
small number of mature, host-responsive T cells could result in small
but significant changes in cytokine production and cause damage in a
sensitive target organ, such as the thymus or the lung, without
producing significant systemic or histologic GVHD in other target
organs. Further studies using genetically marked bone marrow and
thymectomized recipients will be needed to address the relevant
contributions of these two mechanisms to lung damage after allogeneic
BMT.
Enhanced lymphocyte activation in the lungs after BMT has been reported
by others.9,49 Using an experimental BMT model, Gartner and
colleagues49 showed that pulmonary natural killer (NK) cell
activity remained increased over an extended period of time during GVHD
in contrast to the transient and mild increase in splenic NK activity
that occurred during the same interval. Clinically, BAL fluid
lymphocytosis has been described after TCD BMT during pneumonitis that
resulted from a local immune response; pulmonary T cells appeared to be
activated despite systemic immune suppression.9
Furthermore, chronic noninfectious lung injury, as diagnosed by
clinical symptoms, radiographic abnormalities, and alterations in
pulmonary function, has been reported in patients without evidence of
systemic GVHD.31-34,50
Although we have provided data to support a role for alloreactive donor
T cells in the evolution of lung damage after BMT, the precise
mechanisms by which these cells traffic to the lung, interact with host
antigens, and cause injury remain unresolved. This process is likely to
be complex and to ultimately involve the interaction of donor
lymphocytes with pulmonary antigen presenting cells (APC). It is
conceivable that pulmonary dendritic cells, which are potent
stimulators of primary T-cell responses, are intimately involved with
this process.51,52 These cells are thought to play a
critical role in the initiation and regulation of immune responses in
the lung, and recent data suggest that they are important to both acute
and chronic rejection after lung transplantation.53-56
Furthermore, the Th-1 cytokines interleukin-2 (IL-2) and IFN-, which
are critical to the development of GVHD, are felt to be involved in the
activation and recruitment of dendritic cells to sites of
inflammation.57,58 The specific requirement of host
APCs for the generation of acute GVHD was recently
reported in a CD8+ T-cell driven GVHD model where chimeric
animals that did not express MHC class I on their APCs but did express
MHC class I antigens on target tissue were used as BMT
recipients.59 It is possible that radioresistant, pulmonary
APCs in the host persist longer than those in other organs, thus
allowing sustained presentation of host antigens in the lung but not in
other visceral sites to small numbers of donor T cells that are trapped
within the pulmonary microvascular circulation. Studies are in progress
to determine whether the kinetics of dendritic cell turnover after BMT
can account for the apparent "sanctuary" status of the lung with
respect to donor T cells, a concept that may have important
implications with regard to the evaluation and treatment of pulmonary
dysfunction after BMT even when clinical GVHD is absent.
| |
FOOTNOTES |
Submitted March 4, 1998;
accepted June 2, 1998.
Supported by National Institutes of Health Grant Nos. HL55162, CA
39542, and DK39512; J.L.M.F. is a scholar of the Leukemia Society of
America. M.R.M.V. is the recipient of a Howard Hughes Physician
Postdoctoral Fellowship.
Address reprint requests to Kenneth R. Cooke, MD, Dana-Farber Cancer
Institute, Department of Pediatric Oncology, D1420, 44 Binney St,
Boston, MA 02115;e-mail kenneth_cooke{at}dfci.harvard.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract