Download Targeting CD45RB alters T cell migration and delays viral clearance

International Immunology, Vol. 18, No. 2, pp. 291–300
doi:10.1093/intimm/dxh367
ª The Japanese Society for Immunology. 2005. All rights reserved.
For permissions, please e-mail: [email protected]
Targeting CD45RB alters T cell migration
and delays viral clearance
Bock Lim1*, Robyn M. Sutherland2*, Yifan Zhan2, Georgia Deliyannis1, Lorena
E. Brown1 and Andrew M. Lew2
1
Department of Microbiology and Immunology and Cooperative Research Centre for Vaccine Technology,
University of Melbourne, Royal Parade, Melbourne 3010, Australia
2
Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Melbourne 3050, Australia
Keywords: CD45, CTL, homing, influenza, trafficking
Abstract
CD45 is a receptor tyrosine phosphatase essential for TCR signaling. One isoform, CD45RB, is
down-regulated in memory cells and targeting CD45RB with a specific antibody has been shown
to inhibit graft rejection. Its role in immunity to infection, however, has not been tested. Here, we
report the effect of anti-CD45RB antibody treatment on the induction of anti-influenza CD81 T cells
and viral clearance. Anti-CD45RB-treated mice had delayed pulmonary viral clearance compared with
untreated mice whose infection was completely cleared by day 8 post-infection. In anti-CD45RBtreated mice, the total CD41 and CD81 T cell numbers in both the lungs and mediastinal nodes were
substantially reduced at days 5 and 8; this effect was less marked for the spleen. CD81 T cells
specific for influenza virus were also reduced compared with the control group in all three organs at
day 8. By day 11, when both treated and control groups showed no virus remaining in the lungs,
specific CD81 T cell numbers were at similar low levels. Homing to lymph nodes and lung of
dye-labeled T cells was greatly inhibited (by >80%) by anti-CD45RB treatment. This reduced
homing corresponded with reduced CD62L and b1-integrin expression in both uninfected and infected
mice. Since CD62L plays a critical role in homing lymphocytes to lymph nodes, and high levels of
CD62L and a4b1-integrin are expressed by lymphocytes that home to bronchus-associated
lymphoid tissue, we suggest that reduced expression of these molecules is a key explanation for
the delay in immune responses.
Introduction
Cell-mediated adaptive immune responses are critical for the
recovery of primary influenza infection (1). The virus-specific
CD8+ CTLs are thought to be the key effectors in the clearance
of the virus especially in the first 8 days (2) and potentially
provide heterologous immunity by recognizing epitopes that
are highly conserved between different strains of influenza
virus (3). CD4+ T-cell help is relatively dispensable in the
induction of virus clearing CD8+ T cells during a primary
influenza infection (4), although it may be critical for an optimal
CD8+ T cell recall response during secondary infection (5–8).
As we have focused on the primary infection, we have
concentrated our investigations on CD8+ T cells.
CD45 is expressed on all nucleated hematopoietic cells and
is a receptor tyrosine phosphatase critical for antigen-specific
B and T cell responses (9, 10). Due to alternative splicing,
*These authors contributed equally.
CD45 can exist in multiple isoforms (CD45RA, RB, RC and RO)
that can be differentiated by their sizes. Although the
functional relevance of these isoforms is not determined,
they are highly regulated and T cell activation is associated
with a shift from high- to low-molecular weight isoforms. In the
mouse, naive peripheral T cells express abundant CD45RB
and low-level expression is seen on primed/memory T cells, on
cells that show increased secretion of Th2 cytokines, such as
IL-4 and IL-10, and on a population of T cells with regulatory
function (11, 12). These assignments are useful but may be
a slight over-simplification as the CD45 isoform expression is
not necessarily fixed (13, 14).
Studies in transplantation indicate that treatment with the
anti-CD45RB mAb, MB23G2, induces long-term tolerance
to allogeneic grafts in mice (15–18). T cell responses are
Received 4 March 2005, accepted 8 November 2005
Correspondence to: A. M. Lew; E-mail: [email protected]. L. E. Brown; E-mail: [email protected]
Transmitting editor: E. Simpson
Advance Access publication 16 December 2005
292 Anti-CD45RB alters T cell homing and viral clearance
perturbed by this antibody and the cells remaining after antiCD45RB treatment have little or no CD45RB. One interpretation
of these studies could be that the action of the antibody was to
deplete CD45RB cells. However, the possibility of altered
trafficking or sequestration of CD45RB+ cells could not be
discounted. Although anti-CD45RB treatment is not associated
with activation (CD69, CD25 and CD44 are not affected), it is
associated with increased production of both Th2 cytokines
and increased CTLA4 surface expression on CD4+ Tcells (17).
This increase in CTLA4 may be critical in mediating tolerance
(17) and/or reflect a common splice pathway between CD45
and CTLA4 (19).
General immunosuppression by drugs (e.g. cyclosporin,
FK506, rapamycin and mycophenolate mofetil) has been used
clinically to inhibit transplantation rejection successfully;
however, one of the adverse effects of such drugs is increased
susceptibility to serious infections (20, 21). Anti-CD45RB
appears to be less severe than most immunosuppressive
regimens. For example, it does not deplete T cell numbers in
the spleen (17, 22). Surprisingly, the effect of anti-CD45RB
treatment on viral or bacterial immunity has not been reported.
Therefore, we wanted to investigate how such a regimen
would affect the host’s responses to adventitious infections,
e.g. influenza.
Several observations that suggest a link between CD45
expression and trafficking have been reported. Firstly, CD45
has some regulatory role in selectin expression including
CD62L (23) and in integrin-mediated adhesion (24). Secondly,
although anti-CD45RB results in an increase in splenic T cells,
there is also a decrease in circulating lymphocytes (15, 16) and
lymph node T cells (22, 25). Such imbalance in cell numbers
among the various sites could be interpreted as due to
a trafficking aberration. As specific T cells can be more readily
quantified in the murine influenza system than in allogeneic
systems, the model we have chosen also allows trafficking of
antigen-specific T cells to be examined.
In this study, the ability of mice following anti-CD45RB
treatment to induce effective CD8+ T cell-mediated viral
clearing and anti-viral antibody responses during a primary
influenza infection is reported. We also relate this to how T cell
trafficking is affected after anti-CD45RB treatment.
Methods
Mice and anti-CD45RB treatment
C57BL/6 female mice were obtained from the Department of
Microbiology and Immunology, The University of Melbourne
(Parkville, Victoria, Australia). CD4-deficient GK transgenic
mice (26) on a C57BL/6 background were obtained from the
Walter and Eliza Hall Institute (Parkville, Victoria, Australia).
The animals were bred and maintained throughout the
experiment under specific pathogen-free conditions in the
same department. The anti-CD45RB mAb, MB23G2, was
purified on immobilized protein G from hybridoma supernatant. Mice were injected intraperitoneally with 0.2 mg of
MB23G2, 1 day prior to infection and thereafter every 3–4
days. The control mice were injected with either isotype control
(GL117 = rat IgG2a) or PBS.
Virus infection
The type A strain of influenza virus used in this study was
a genetic reassortant of A/Memphis/1/71 (H3N2) 3 A/Bellamy/
42 (H1N1) referred to as Mem71 (H3N1) virus. The split
Mem71 virus for use in ELISA was prepared by rate zonal
centrifugation to purify the virus before inactivation with
(b-propiolactone and disruption with sodium taurodeoxycholate. Mice were infected intranasally with 104.5 plaque-forming
units of Mem71 virus after being anesthetized with penthrane.
Each mouse received 50 ll of the virus in the form of allantoic
fluid diluted in RPMI-1640.
ELISPOT (Enzyme linked immunospot) assay for
IFN-c-secreting CD8+ T cells
Influenza-specific IFN-c-secreting CD8+ T cells were enumerated by an ELISPOT assay as previously described (27) with
2 lg ml1 of a peptide representing the dominant H-2Dbrestricted epitope NP366–374, ASNENMETM (28).
Flow cytometric analysis of lymphocytes
Lungs were minced, digested in 2 mg ml1 collagenase A
(Roche, Mannheim, Germany) for 30 min at 37C, and then
dispersed through a metal sieve. The resultant cell suspensions were pelleted and washed. Liver lymphocytes were
obtained as described (29). Briefly, livers were minced and
pushed through a metal sieve. Hepatocytes were removed by
centrifugation in a 36% solution of isotonic Percoll in PBS, and
the pellet containing lymphomyeloid cells and RBC was
recovered. RBCs were removed from suspensions of liver,
lung and spleen cells by lysis in Tris-buffered ammonium
chloride solution. In experiments where liver lymphocytes
were analyzed, mice were perfused by injecting 20 ml of PBS
into the heart prior to recovery of organs.
CD8+ and CD4+ T cells were enumerated by flow cytometry
using a FACScan (Becton Dickinson, San Jose, CA, USA) after
staining the lymphocytes with FITC-conjugated rat anti-mouse
CD8 antibody (clone 53-6.7, Pharmingen, San Diego, CA,
USA) and PE-conjugated rat anti-mouse CD4 antibody (clone
GK1.5, Pharmingen). Data were analyzed using FlowJo
version 3.6.1 (Tree Star Inc., San Carlos, CA, USA). Expression
of b7-integrin and L-selectin on CD8+ T cells was examined by
co-staining with PE-conjugated anti-CD8 antibody (clone
CT-CD8a, Caltag Laboratories, Burlingame, CA, USA) and either
FITC-conjugated anti-b7-integrin antibody (Pharmingen, clone
M293) or FITC-conjugated anti-L-selectin antibody (clone
MEL-14, Pharmingen). Expression of b1-integrin on CD8+
T cells was examined by co-staining with PE-Cy5-conjugated
anti-CD8 antibody (clone 53-6.7, Pharmingen) plus FITCconjugated anti-b1-integrin antibody (clone Ha2/5, Pharmingen). Expression of CD45RB was examined with a biotinylated
mAb that did not compete with MB23G2 (clone 16A,
Pharmingen). Propidium iodide staining was used to exclude
dead cells. Cells were analyzed using a FACScan and
CellQuest software.
ELISA
To determine the titer of influenza-specific total and isotype
(IgM, IgG1, IgG2c and IgG2b) antibodies in the sera of
influenza-infected mice, flat-bottom 96-well polyvinyl chloride
Anti-CD45RB alters T cell homing and viral clearance 293
microtiter plates (Dynatech) were coated with 50 ll per well of
split Mem71 virus at a concentration of 5 mg ml1 in PBSN3 (30).
The secondary antibodies used were 1:400 dilution of HRPconjugated rabbit anti-mouse antibodies (DakoCytomation,
Carpinteria, CA, USA) or 1:5000 dilution of HRP-conjugated
goat anti-mouse isotyping antibodies (Southern Biotechnology
Associates Inc., Birmingham, AL, USA). The substrate used
was 0.2 mM of 2-29-azino-bis(3-ethylbenzthiazoline-6-sulfonic
acid) (Sigma Chemicals Co.) in 50 mM citrate buffer, pH 4.0,
containing 0.004% H2O2 (v/v). The reaction was stopped by
adding 50 ll of 45 mM sodium fluoride (Sigma Chemicals Co.)
to each well. Plates were read at the dual wavelength of 405 and
450 nm (Multiskan Multisoft Labsystems, Helsinki, Finland). The
antibody titer was expressed as the reciprocal of dilution of
serum giving an optical density five times higher than the
background with normal mouse serum.
Plaque assay
Infectious virus was quantified by plaque assay conducted on
confluent monolayers of Madin–Darby canine kidney cells as
previously described (30, 31).
Results
Clearance of influenza virus in mice after anti-CD45RB
treatment
To investigate the effect of anti-CD45RB on anti-influenza
immunity that leads to viral clearance in the lungs, mice were
treated intraperitoneally with 0.2 mg of anti-CD45RB 1 day
prior to intranasal infection of influenza virus, and every 3 days
thereafter. Control mice were either injected with PBS or the
control isotype GL117; both controls behaved similarly. The
ability of these mice to clear the pulmonary virus was examined
5, 8 and 11 days post-infection by plaque assay on samples
from the lungs. On day 5, prior to the recruitment of CD8+
effector T cells to the lungs, viral titers were high in both groups
(Fig. 1). By day 8, control mice had completely cleared the
virus in their lungs (Fig. 1). In contrast, mice treated with antiCD45RB showed a delayed viral clearance, with individual
mice displaying a range of viral titers in their lungs. All the
treated mice ultimately cleared the virus by day 11 postinfection. Thus, anti-CD45RB hindered but did not abrogate
the ability of the mice to clear a pulmonary influenza infection.
Homing of CD8+ T cells labeled with carboxyfluorescein
diacetate succinimidyl ester after anti-CD45RB treatment
Expansion of lymphocytes in the mice following
influenza infection
A single-cell suspension was made from inguinal, axillary,
popliteal, renal, iliac and mediastinal lymph nodes (MLN)
recovered from C57BL/6 mice. The cells were washed twice
with 0.1% BSA (CSL Ltd) in PBS before labeling with 10 lM
5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE,
Molecular Probes Inc., Eugene, OR, USA). After 10 min at
37C in the presence of CFSE, the cells were washed twice
with medium and twice with PBS. CFSE-labeled cells (1.3 3
107) in PBS were injected intravenously into each C57BL/6
mouse, followed immediately by either MB23G2 or PBS
delivered intraperitoneally. The mice were infected on the
following day with Mem71. On day 5 post-infection, spleen,
liver, lung and MLN and inguinal lymph nodes (ILN) were
harvested from mice. The total number of CFSE-labeled CD8+
T cells in the various organs was determined by flow cytometry
after staining with PE-conjugated rat anti-mouse CD8 antibodies (clone 53-6.7, Pharmingen).
During infection, the rapid proliferation of lymphocytes is
important for an efficient recovery from pulmonary influenza.
Here, in an attempt to explain the delayed viral clearance in
mice treated with anti-CD45RB, the expansion of the lymphocytes in response to the infection was examined. The total
numbers of CD4+ T cells and CD8+ T cells in the MLN, lungs
and spleen of mice on days 5, 8 and 11 are shown in Fig. 2. On
day 5 post-infection, in the MLN and lungs, the total numbers
of lymphocytes were significantly higher for control mice than
for anti-CD45RB-treated mice. This early expansion of
lymphocytes in the control mice is consistent with their ability
to completely clear virus within the next 3 days. As expected,
on day 8 post-infection, control mice that had just cleared the
virus had very high numbers of CD4+ and CD8+ lymphocytes
in the lung and MLN and these levels had decreased by day
11 in the absence of further stimulus. The rise in T cell numbers
on day 8 post-infection was particularly notable in the lungs
In vitro treatment of CD8 T cells with anti-CD45RB
Pooled lymph node cells from the ILN, axilliary, popliteal and
mesenteric lymph nodes of CD4 T cell-deficient GK mice were
enriched for CD8 T cells by passing twice over nylon wool
columns. Cells (4 3 106) in 1 ml of RPMI supplemented with
10% (v/v) heat-inactivated FCS and 50 lM 2-mercaptoethanol
were added to a 24-well tissue culture plate. Either MB23G2
anti-CD45RB or GL117 isotype control antibody was added at
a final concentration of 0.2 lg ml1 and cultures were
incubated for 22 h at 37C. At the conclusion of culture, cells
were recovered for FACS analysis.
Statistical analysis
Statistical analysis was performed using the non-parametric
Mann–Whitney U test on the Prism 4 software program
(GraphPad Software Inc., San Diego, CA, USA).
Fig. 1. Anti-CD45RB treatment delays virus clearance. Influenza virus
load in the lungs of control and anti-CD45RB-treated mice was
determined on days 5, 8 and 11 after intranasal infection. The geometric
mean titer of a group is shown as a line, and each symbol represents an
individual mouse. Log10 1.6 is the minimum detectable titer.
294 Anti-CD45RB alters T cell homing and viral clearance
Fig. 2. The total number of T cells in lymph nodes and lungs can be reduced by anti-CD45RB treatment. Total numbers of CD4+ and CD8+ T cells
recovered from the MLN, lung and spleen of control and anti-CD45RB-treated mice on days 5, 8 and 11 post-infection. The mean is shown as
a line, and each symbol represents an individual mouse.
where the viral clearing activity was taking place (CD4+ T cells,
P = 0.008; CD8+ T cells, P = 0.008). When the mice were
treated with anti-CD45RB, fewer T cells were present in both
MLN and lung. This deficit was particularly dramatic in the
MLN (day 8: CD4+ T cells, P = 0.008; CD8+ T cells, P = 0.016
and day 11: CD4+ T cells, P = 0.016; CD8+ T cells, P = 0.008).
In contrast to the MLN and lung, the difference in T cell
numbers in the spleens between anti-CD45RB-treated and
untreated mice through most of the study was unremarkable.
Only for the CD8+ T cells and only on day 8 post-infection
(when there was also a maximum increase in antigen-specific
CD8+ T cells; Fig. 3) was there a significant difference. The
general lack of changes in total T cell numbers in the spleen is
consistent with recent reports that anti-CD45RB treatment did
not decrease the splenic T cell numbers (17, 22). This modest
effect on spleen compared with lymph nodes was one of the
early indications that there may be altered trafficking.
Influenza-specific CD8+ T cell responses in the mice
Early control of primary influenza infection involves CD8+
T cell-mediated immunity. Influenza-specific IFN-c-secreting
CD8+ T cells were enumerated in the MLN, lungs and spleen of
the mice on the various days after viral infection using an
ELISPOTassay (Fig. 3). The numbers of antigen-specific CD8+
T cells increased considerably between day 5 and day 8 postinfection in all organs. On day 8, the control mice had an
abundance of influenza-specific IFN-c-secreting CD8+ T cells
in their MLN, lungs and spleen, correlating with their ability to
clear the virus (Fig. 1). However, mice treated with antiCD45RB had significantly fewer influenza-specific IFN-csecreting CD8+ T cells in the MLN (P = 0.008), lungs (P =
0.032) and spleen (P = 0.008) as compared with the controls.
These data parallel the reduced total CD8+ T cell numbers
(Fig. 2) in the MLN and lungs of anti-CD45RB-treated mice.
Nevertheless, influenza-specific IFN-c-secreting CD8+ T cells
were present in the virus-infected lungs of all the mice. In order
to have an estimate of what proportion of CD8+ T cells was
activated to secrete IFN-c, we divided the ELISPOT numbers
in Fig. 3 by the total CD8+ Tcell numbers in Fig. 2 (Table 1). The
numbers are so low on day 5 that they essentially correspond
to uninfected mice. There were no statistical differences in the
proportion of specific T cells to total CD8+ T cells for treated
Anti-CD45RB alters T cell homing and viral clearance 295
compared with control mice in the various organs on day 8. By
day 11 in the MLN, this proportion was actually higher for antiCD45RB-treated mice (11% versus 1%; P < 0.01), even
though the total numbers of influenza-specific cells in the
organ were similar between treated and control mice (Fig. 3).
The finding that total CD8+ T cell numbers were dramatically
reduced by anti-CD45RB without a substantial decrease in the
proportion of specific versus total CD8+ T cells argues against
any major effect of anti-CD45RB on T cell activation, but would
be consistent with a lack of recruitment.
By day 11 post-infection, the numbers of influenza-specific
IFN-c-secreting CD8+ T cells in the lungs in untreated mice
were greatly reduced, as the immune response subsided after
viral clearance (Fig. 1). In the anti-CD45RB-treated mice, there
was a modest increase of anti-viral CD8+ T cells such that on
day 11 there were more CD8+ T cells in the anti-CD45RB mice
than the controls. This could be due in part to the delayed
kinetics of the immune response in anti-CD45RB-treated mice,
but could also be in part due to the infection lasting beyond
day 8 in these mice. In conclusion, the observed diminished
CD8+ T cell response after anti-CD45RB treatment on day 8
post-infection correlates with the delayed viral clearance in
these mice. However, all anti-CD45RB-treated mice had
ultimately cleared the pulmonary influenza by day 11 postinfection, despite the lack of a strong peak in CD8+ T cell
number as observed in control mice on day 8 post-infection.
Anti-influenza humoral response in the mice
Fig. 3. Anti-CD45RB treatment reduces total influenza-specific CD8+
T cells. Influenza-specific IFN-c-secreting CD8+ T cells in the MLN,
lung and spleen of control and anti-CD45RB-treated mice on days 5, 8
and 11 post-infection were enumerated by ELISPOT assay and
expressed as numbers per organ. Error bars represent standard
deviation of the mean.
The developing antibody response is known to be important in
aiding recovery at later time points after infection. Since CD45
is known to play a role in antigen-specific B cell responses,
treatment with anti-CD45RB might affect the humoral response
against the infection, which in turn could influence the level of
viral clearance. Hence, the total anti-influenza antibody in the
sera of these mice was determined using ELISA and the
results are illustrated in Fig. 4. On day 5 post-infection, no
influenza-specific antibody was detected in either group of
mice. On day 8 post-infection, mice treated with anti-CD45RB
had significantly less antibody against influenza in their sera
compared with the untreated controls. However, on day 11
post-infection, the total anti-influenza antibody titers in the
anti-CD45RB-treated mice had risen dramatically (10-fold
increase; P = 0.008). The profiles of IgM, IgG1, IgG2b and
IgG2c (32) isotypes within these sera were also examined
but no significant differences between the anti-CD45RBtreated mice and PBS-treated controls were apparent (data
Table 1. Number of ELISPOTs per 100 CD8+ T cells MLN, lungs and spleens of mice treated with anti-CD45RB as compared with
controls (n ¼ 6)
Post-infection
Treatment
MLN
5 days
PBS
Anti-CD45RB
PBS
Anti-CD45RB
PBS
Anti-CD45RB
0.2
0.1
1.1
0.6
0.7
11.3
8 days
11 days
Lung
6
6
6
6
6
6
0.1
0.1 (P ¼ 0.02)
0.5
0.5 (P ¼ 0.10)
0.4
6.6 (P ¼ 0.01)
2.2
1.2
63.5
131.4
38.2
51.6
Spleen
6
6
6
6
6
6
2.8
1.7 (P ¼ 0.55)
33.1
102.7 (P ¼ 0.22)
38.5
27.6 (P ¼ 0.31)
0.02
0.03
1.1
0.7
0.4
0.3
6
6
6
6
6
6
0.01
0.02 (P ¼ 0.31)
0.6
0.3 (P ¼ 0.31)
0.2
0.2 (P ¼ 0.84)
296 Anti-CD45RB alters T cell homing and viral clearance
Fig. 4. Anti-CD45RB treatment delays the humoral response. Total
influenza-specific antibody was determined by ELISA. Sera from
control and anti-CD45RB-treated mice were collected on days 5, 8 and
11 post-infection and compared with normal mouse serum (nms). The
geometric mean titer and standard deviation are indicated.
not shown). Taken together, these data propose that antiCD45RB treatment delays the induction of the humoral response against influenza virus and suggest a possible role
of influenza-specific antibodies in the clearance of virus observed in the anti-CD45RB-treated mice on day 11 postinfection in the absence of a strong CD8+ T cell response.
Altered T cell homing after anti-CD45RB treatment
We have shown above that the numbers of lymphocytes
(especially CD8+ T cells) that gather at the site of infection are
dramatically reduced. The lymphocytes that home to the
bronchus-associated lymphoid tissue have high levels of
CD62L and a4b1-integrin on their surface (33), whereas guthoming lymphocytes bear high levels of a4b7-integrin (34).
However, there is no such preference in the spleen, so we
decided to investigate the effect of anti-CD45RB antibody
treatment on the homing molecules CD62L (L-selectin), b7 and
b1 on CD8+ T cells in this organ. On day 8 after infection, antiCD45RB treatment resulted in far fewer cells bearing high
levels of these homing molecules: for CD62L from 66 6 5% to
39 6 4% and for b1-integrin from 35 6 3% to 22 6 7% (Fig. 5).
In uninfected mice (n = 3), the reduction was, respectively,
from 87 6 3% to 44 6 4% and from 12 6 1% to 9 6 1%.
Interestingly, cells with high levels of b7-integrin are also
reduced, indicating that perhaps homing to the gut might also
have been altered. The effect of the anti-CD45RB treatment on
homing molecules was not simply due to deletion of CD45RB
cells. Firstly, there was little reduction of CD45RBhi cells in the
spleen (data not shown). Even in the lymph nodes, there was a
substantial proportion (31.6%) of CD45RB+ cells remaining
after anti-CD45RB treatment versus the 67.8% in control mice
(Fig. 6). In contrast, only 11.9% of cells remained CD62L+
(Fig. 6) and therefore most CD45RB+ cells must have downmodulated their CD62L.
To determine the effect of anti-CD45RB antibody on
homing more directly, we tracked dye-labeled CD8+ T cells
in vivo. CD8+ T cells from naive mice were labeled with the dye
CFSE and injected intravenously. The numbers of CFSElabeled cells that tracked to the spleen, lung, draining MLN,
Fig. 5. Anti-CD45RB treatment reduces the percentage of CD8+
T cells bearing high levels of CD62L or b1- or b7-integrins. Spleen cells
were recovered from control and anti-CD45RB-treated mice 8 days
post-infection. Representative plots are shown for one of three mice in
each treatment group, and are gated on viable CD8+ T cells.
Fig. 6. Anti-CD45RB treatment reduces CD62L expression on
lymphocytes expressing high levels of CD45RB. Lymph nodes were
recovered 3 days after treatment with control isotype or anti-CD45RB
antibody. CD45RB staining was performed using the 16A antiCD45RB antibody which does not compete with the MB23G2 antiCD45RB antibody used for in vivo treatment. Plots are gated on viable
lymphocytes.
Anti-CD45RB alters T cell homing and viral clearance 297
non-draining ILN and liver all decreased with anti-CD45RB
treatment (Fig. 7). Although the untreated donor CD8 T cells
appeared homogeneous with respect to expression of
CD45RB and CD62L (Fig. 7a), treatment with anti-CD45RB
antibody preferentially reduced their ability to traffic to lungs
and lymph node compared with spleen. Thus, in infected
mice, anti-CD45RB treatment reduced trafficking to the spleen
by 53% compared with a reduction in excess of 80% in lungs
and lymph node (lung, 84%; MLN, 81%; ILN, 90%; Fig. 7b).
Similarly, in uninfected mice, there was a 69% reduction in
spleen compared with a reduction of at least 87% in lungs and
lymph node (lung, 87%; MLN, 95%; ILN, 97%; Fig. 7b).
Anti-CD45RB treatment directly reduces expression of
CD62L on CD8 T cells
The overall reduction in CD8 T cell recovery in anti-CD45RBtreated mice (Fig. 7) indicated that this antibody can deplete
CD8 T cells or results in their being sequestered in an as yet
unidentified site. Thus, it was important to determine if antiCD45RB simply depletes/sequesters naive CD8+T cells
expressing high levels of CD62L/CD45RB or is also able to
directly affect expression of CD62L. CD62L levels were clearly
decreased upon in vitro incubation of purified CD8 T cells with
anti-CD45RB antibody (Fig. 8). Notably, a concurrent decrease
in CD45RB expression was not observed (Fig. 8).
Discussion
The effect of the anti-CD45RB mAb, MB23G2, on primary
influenza infection was studied here. Virus-specific CD8+
Fig. 7. Anti-CD45RB treatment dramatically reduces CD8+ T cell
trafficking to lymph nodes and lung. Lymph node cells were harvested
from naive syngeneic female donors and analyzed for CD62L and
CD45RB expression on CD8 cells (Fig. 7a). The donor cells were then
labeled with CFSE dye and adoptively transferred to mice immediately
prior to either anti-CD45RB or isotype control antibody treatment. The
mice were infected on the following day. The spleen, lungs, MLN, ILN
and liver of recipient mice were recovered on day 6 post-transfer and
analyzed by flow cytometry. The number of CFSE-labeled CD8+ T cells
in anti-CD45RB mice was expressed as a percentage of the number in
isotype control–treated mice. The means and standard deviations of
absolute numbers in isotype control–treated mice were as follows—infected: spleen, 57 269 (19 324); lung, 1882 (747); MLN, 4740 (3128);
ILN, 4667 (407), and liver, 472 (120); and uninfected: spleen, 65 091
(11 337); lung, 1032 (334); MLN, 90 (52); ILN, 19 096 (14 854), and
liver, 743 (352).
Fig. 8. In vitro treatment with anti-CD45RB antibody results in decreased expression of CD62L on CD8 T cells. Purified CD8 T cells were
incubated with 0.2 lg ml1 of either MB23G2 anti-CD45RB (open
histogram) or GL117 isotype control (closed histogram) antibody for
22 h. FACS analysis was performed at the conclusion of culture. Data
shown for CD8+propidium iodide gated lymphocytes.
298 Anti-CD45RB alters T cell homing and viral clearance
T cells are known to be critical for the recovery of the early
phase of influenza infection (2). In the murine influenza
pneumonia model at the time of virus clearance, CD8+ T cells
dominate the cellular population in the bronchoalveolar lavage
(35). Mice that were treated with anti-CD45RB demonstrated
a delayed viral clearance response in which significant viral
load could be detected in the lungs of these mice on day 8 postinfection, while the control mice had completely cleared the
pulmonary influenza. This delayed clearance corresponded
with the significant reduction in numbers of influenza-specific
CD8+ T cells in the lungs and MLN of the anti-CD45RB-treated
mice.
Our findings suggest that the anti-influenza-specific CD8+
T cell response leading to viral clearance is altered. This is
concordant with studies that reported the perturbation of T cell
response after anti-CD45RB treatment in allograft models
(15, 16). The most marked effect of anti-CD45RB treatment in
our study was the reduction in lymphocyte numbers in the
MLN. In contrast, in the spleen, numbers were either unaltered
or modestly reduced (Fig. 2). This resulted also in reduced
numbers of influenza-specific CD8+ T cells in the whole MLN,
but as a percentage of CD8+ T cells, there was no significant
reduction (Table 1). This hinted that cell activation per se was
not markedly affected by anti-CD45RB treatment and that the
effect may be due to reduced naive cell trafficking to the
regional lymph node for priming. For confirmation, we showed
that the numbers of lymphocytes with high levels of the
adhesion molecules associated with homing to lymph nodes
were reduced (Fig. 5). The homing molecules we considered,
L-selectin, a4b1 and a4b7, are important for homing from the
blood to the high endothelial vessels of MLN (33); the latter two
are also important in trafficking to the lungs (36). Less is known
about what adhesion molecules are required (if any) for
lymphocytes from afferent lymph; however, they comprise only
10% of lymphocytes entering lymph nodes, whereas blood
lymphocytes comprise >85% (37). We contend that the
reduction in cells expressing these homing molecules is not
merely due to depletion/sequestration of naive cells. Firstly,
there is very little depletion in the spleen. Secondly, the
moderate reduction in numbers of CD45RB+ cells in lymph
nodes cannot explain the dramatic reduction in numbers of
CD62L+ cells (Fig. 6). Thirdly, CD62L expression was reduced
under conditions where neither preferential depletion nor
sequestration of CD45RB-high cells occurred viz. in vitro
incubation with anti-CD45RB antibody (Fig. 8). Fourthly, the
anti-CD45RB antibody used is a rat IgG2a and this isotype is
inefficient at depletion (38), probably because its hinge region
does not contain the Leu-Leu-Gly-Gly-Pro motif (39).
As well as the surface phenotype studies, functional studies
of homing by CFSE-labeled lymphocytes (Fig. 7) showed that
the greatest reduction of homing was to the lymph node after
anti-CD45RB treatment. The impaired recruitment of naive
cells entering the nodes thus explains the dramatic decrease
in CD4+ and CD8+ T cell numbers in the MLN of the antiCD45RB-treated mice (Fig. 2). This in turn would lead to
reduced numbers of specific T cells being primed and hence
(at least in part) delayed viral clearance. In support of our
findings, a single dose of anti-L-selectin antibody treatment
has been shown to delay the response to Sendai virus (40).
Interestingly, mice that transgenically expressed only CD45RO
did not have any alteration in lymphocyte distribution and
were able to clear influenza infection by day 9 (41). We
assume that some compensation has happened during
lymphocyte development that allowed normal homing.
Lymphocyte trafficking as a modality for controlling immune
responses has received much attention recently, especially
with the uncovering of the molecular basis of the immunosuppressive drug FTY720 (42, 43). This drug blocks egress of
lymphocytes from lymph nodes by its action on the sphingosine1-phosphate 1 receptor. In contrast to egress, we have shown
that anti-CD45RB antibody treatment results in reduced ingress of lymphocytes to lymph nodes. We therefore speculate
that the combination of FTY720 and anti-CD45RB would have
an even more dramatic effect on immune responses.
Interestingly, by day 11 post-infection, mice treated with
anti-CD45RB ultimately cleared the pulmonary infection.
Although the homing mechanism to the draining MLN of the
lungs appears affected after anti-CD45RB treatment, the
suboptimal CD8+ T cell response that was observed in these
animals could have been mostly induced in the spleen (44).
This splenic population of activated CD8+ T cells might be
sufficient to generate CTL effectors needed for viral clearance
eventually. This is because the optimal CD8+ T cell response
seen in control mice is probably in excess and a more modest
response might be able to ultimately control the pulmonary
influenza (45). The increase of anti-influenza antibody in antiCD45RB-treated mice on day 11 post-infection could also aid
in viral clearance, as the humoral response is also known to
play a role in the recovery of influenza (46, 47).
The effect of anti-CD45RB treatment on the humoral
response against the influenza infection was also analyzed
here. Mice treated with anti-CD45RB showed a delayed
humoral response on day 8 post-infection but total antiinfluenza antibody titer increased dramatically by day 11 postinfection. Though CD45 is known to play a role in B cell
activation by mediating signals via the B cell antigen receptor
(9), the mechanism in which anti-CD45RB treatment affects
the humoral response is not well studied. As B cells share
many of the homing receptors of T cells (e.g. CD62L, a4b1),
the treatment of anti-CD45RB could also have impaired the
trafficking of B cells and resulted in the delayed humoral
response seen. We did not find significant differences in the
isotypes of the total anti-influenza antibody between the antiCD45RB-treated mice and the controls, even though antiCD45RB treatment has been shown to enhance the production
of Th2 cytokines (15, 25).
Interestingly, treatment of anti-CD45RB has been reported
to show greater immunosuppression and enhanced graft
tolerance when accompanied with the blockade of B7/CD28
and CD40/CD40L co-stimulatory pathways (22, 25). Moreover,
anti-CD45RB treatment is shown to up-regulate CTLA4
expression on T cells that block B7/CD28 co-stimulation (17).
To prevent allograft rejection in clinical transplantation,
immunosuppression is mediated usually by a combination of
drugs (e.g. FK506, mycophenolate mofetil) that are broad
spectrum. As such, one of the adverse effects is increased
susceptibility to infections (20, 21). Thus, one of the main aims
in transplantation is to have more selective immunosuppression and anti-CD45RB may be a candidate. If anti-CD45RB
were ever to be used clinically in transplant patients, it would
Anti-CD45RB alters T cell homing and viral clearance 299
be useful to know whether the host could resist infections.
We have shown here that although influenza clearance was
slower (by up to 3 days), the host was still able to eliminate
the infection. Thus, anti-CD45RB may be a more strategic form
of immunosuppression than the current drugs employed.
The treatment of anti-CD45RB has been demonstrated here
to impede viral clearance of pulmonary influenza by affecting
the induction of an optimal CD8+ T cell response and causing
a delay in the humoral response. Although the pulmonary
infection was eventually cured in the absence of an optimal
induction of CD8+ T cell response, this should be seen in the
light of primary influenza infection. Recent experiments have
shown that optimal induction of memory CD8+ T cells requires
the interplay of various immune components such as CD4+
T cells and appropriate co-stimulation (5, 6, 8, 48, 49). This is
vital for vaccination schemes to provide long-term protection.
Thus, it will be interesting to examine the effect of anti-CD45RB
treatment on secondary viral infection, although long-term
studies with the anti-CD45RB mAb would be difficult, as mice
eventually make antibody against the rat mAb rendering it
ineffective.
The insights on the roles of CD45RB in the induction of
immune response will define important parameters in both
transplantation and vaccination. In summary, this study indicates that CD45RBhi cells are critical in the optimal induction of
influenza-specific CD8+ T cells response but are not mandatory for viral clearance.
Acknowledgements
This work was supported by Juvenile Diabetes Research Foundation,
National Health and Medical Research Council of Australia and
the Australian Government via its Cooperative Research Centres
Program. B.L. is a recipient of the Australian Postgraduate Award.
Abbreviations
CFSE
ILN
MLN
5,6-carboxyfluorescein diacetate succinimidyl ester
inguinal lymph nodes
mediastinal lymph nodes
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