2057
The Journal of Experimental Biology 200, 2057–2062 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
JEB0921
TRAFFICKING OF ADOPTIVELY TRANSFERRED B LYMPHOCYTES IN
B-LYMPHOCYTE-DEFICIENT MICE
RITA ROTH AND MARK J. MAMULA*
Section of Rheumatology, Yale University School of Medicine, New Haven, CT 06520-8031, USA
Accepted 23 May 1997
Summary
intraperitoneal and intravenous injection, B cells were
Many studies have investigated the fate of adoptively
found in approximately equal numbers in the lymph nodes
transferred lymphocytes in recipient mice, although little
and the spleen. Two days later, the B-cell distribution in
is known of the sites where these transferred cells reside at
the lymphoid organs appeared to be independent of the
particular time points. Using flow cytometry, we analyzed
mode of B-cell transfer. A transient decrease in the
the trafficking pattern of adoptively transferred naive B
numbers of splenic and lymph node B cells occurred 9 days
cells into the lymphoid organs of syngeneic B-cell-deficient
µMT) mice. Within the first 24 h of transfer, the location
after B-cell transfer (a decrease from 70 to 87 %) prior to
(µ
of B cells was highly dependent on the mode of B-cell
the outgrowth of B cells that occurs 21 days after transfer.
transfer. When B cells were injected subcutaneously into
These studies are useful for understanding the numbers of
µMT mice, they showed a different trafficking pattern
B cells that may be required in adoptive transfer studies
from cells administered into the peritoneal cavity or
and their potential cellular interactions at particular
injected intravenously. After subcutaneous transfer into
physiological sites based on the route of cell transfer.
the thigh, the greatest number of B cells was detected in the
popliteal lymph node nearest to the injection site, whereas
the lowest number was detected in the axillary lymph node
Key words: B-lymphocyte trafficking, B-cell circulation, adoptive
transfer, mouse.
opposite to the injection side. Within the first 24 h of either
Introduction
B lymphocytes recirculate in the body along defined routes
as they patrol the body in search of invading antigen. Bonemarrow-derived naive B cells enter secondary lymphoid
organs from the blood and migrate through the T-cell rich
paracortical area into primary follicles, where they remain for
up to 24 h before their recirculation. The B cells then circulate
continuously between the blood and lymphoid tissues (Gowans
and McGregor, 1965; Howard, 1972; Gutman and Weissman,
1973; Niewenhuis and Ford, 1976; Picker and Butcher, 1992).
The circulating B-cell pool of a healthy mouse consists of
approximately 200 million cells, most of which survive for
more than 4 weeks (Sprent and Miller, 1972; Gray 1988;
Forster and Rajewsky, 1990). The trafficking patterns and
homing of lymphocytes are dictated by specific molecular
signals which are responsible for the preferential recirculation
patterns of distinct lymphocyte subpopulations (reviewed in
Picker and Butcher, 1992; Springer, 1994).
One approach to studying the trafficking of B cells has been
by the transfer of radioactively labeled B cells into B-cell
depleted (anti-µ-treated) mice (Ron and Sprent, 1987). The
recent development of B-cell-deficient (µMT) mice by gene
*Author for correspondence (e-mail: [email protected]).
targeting (Kitamura et al. 1991) allows the investigation of Bcell trafficking in an environment free of interfering anti-µ
antibody and without the need for B-cell prelabeling. In this
study, we analyzed the distribution of splenic B cells from
syngeneic donor mice into the lymphoid organs of µMT mice
following different modes of cell transfer. The identification
and quantification of B cells were performed using flow
cytometry. Our study shows that the initial distribution of the
B cells is highly dependent on the mode of cell transfer.
Analysis of recipient mice at later times demonstrated that the
population of B cells in peripheral lymphoid organs was less
dependent on the route of adoptive transfer. Finally, the
recipient mice demonstrate a transient decrease in overall Bcell numbers prior to the outgrowth of adoptively transferred
cells.
Materials and methods
Animals and B-cell adoptive transfer
Female B10.A mice, 6–8 weeks of age (NIH, Bethesda,
MD), were used as B-cell donors. B-cell-deficient mice (µMT
2058 R. ROTH
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M. J. MAMULA
mice; a kind gift of Dr C. A. Janeway, Jr) were generated by
disrupting the µ chain constant region of IgM (Kitamura et al.
1991). µMT mice were backcrossed (tenth generation) and
maintained on the B10.A background in the animal care
facilities at Yale University. Mice made homozygous for the
µMT mutation lack B cells expressing B220, sIgM or sIgD (see
Fig. 1 and data not shown) and have no detectable serum IgM
(data not shown). These mice, at 8–10 weeks of age, served as
B-cell recipients. Purified splenic B cells (15×106 cells) in
100 µl of phosphate-buffered saline (PBS) from wild-type mice
were subcutaneously injected into the left lower thigh,
administered into the peritoneal cavity or intravenously
administered into the tail vein of recipient µMT mice. All mice
were used in accordance with approved procedures from the
Yale University School of Medicine, Division of Animal Care.
Yale University is a registered research facility with the US
Department of Agriculture in compliance with the American
Association for Accreditation of Laboratory Animal Care.
complement-mediated cell purifications were: anti-Thy-1
(Y19; Jones, 1983), anti-CD4 (GK1.5; Dialynas et al. 1983),
anti-CD8 (TIB105 and TIB 210) and anti-Mac1 (TIB 128).
FITC-conjugated anti-B220 monoclonal antibody (mAb)
(RA3-6B2) (Coffman and Weissman, 1981) and FITCconjugated rat IgG2a were purchased from Pharmingen (San
Diego, CA). Flow cytometry staining was performed with
5×105 cells in 50 µl of PBS, 0.1 % bovine serum albumin
(BSA) and 0.02 % sodium azide in U-shaped microtiter plates.
Culture supernatants of monoclonal antibodies were used
undiluted in incubations with cells. FITC-conjugated
antibodies were used according to the manufacturer’s
instructions. Test samples and isotype controls were analyzed
on a FACScan instrument (Becton-Dickinson, Mountain View,
CA).
Preparation of donor B cells
B-cell suspensions from wild-type B10.A mice were
prepared by gentle disruption of the spleen between frosted
glass slides. Erythrocytes were depleted and the remaining
cells were washed with Click’s EHAA medium (Irvine
Antibodies and flow cytometry
The antibodies utilized in flow cytometric analysis and
A
106
Relative cell number
100
7.5×105
4.5×105
50
3×105
0
0
100
101
102
50
105
102 103 102 103 102 101 102 103 102 103
Fluorescence intensity
B
Fig. 1. Standard curve for the detection and semi-quantitative analysis
of B cells. (A) Lymph node cells (LNCs; 0.5×106) from µMT (B-celldeficient) mice were mixed with increasing numbers of B cells, as
indicated. Cell fractions were stained with anti-B220 antibody and
analyzed by flow cytometry. The histogram on the left shows the
staining of µMT LNCs lacking B cells. The individual peaks were
spliced into a single figure and represent the B-cell staining with antiB220–FITC obtained from LN cell fractions titrated with B cells (105
to 106). (B) Relative cell numbers taken from A represent the fraction
of B cells among total LNCs analyzed (y-axis). These percentages are
plotted against the absolute number of B cells added on the y-axes.
The results are the means of six titration experiments. Standard
deviations were less than 8 %.
Relative cell number (%)
40
30
20
10
0
0
2×105
4×105 6×105
B cell number
8×105
106
B lymphocyte trafficking 2059
a million cells from µMT mice were supplemented with
increasing numbers of purified B cells from wild-type B10.A
mice (0–106 B cells). The cell populations were then stained
with anti-B220–FITC. As illustrated in Fig. 1A, flow cytometry
could clearly detect 105 cells within stained populations of
LNCs. Fig. 1B shows the signal intensity of the added B cells
(as determined in Fig. 1A) plotted as a percentage of total LNCs
in the staining mixture. The lower limit of detection is
approximately 0.5×105 B cells (as plotted in Fig. 1B). The
standard curve is linear (correlation coefficient 0.997) up to
7.5×105 added B cells, where the signal reaches a plateau. This
information allows for a semi-quantitative analysis of B cell
numbers among LNCs in experimental mice.
Scientific, Santa Ana, CA, USA) supplemented with
2 mmol l−1 L-glutamine, 0.1 mmol l−1 β-mercaptoethanol,
100 u ml−1 penicillin and 100 µg ml−1 streptomycin. Cells were
incubated with a cocktail of undiluted culture supernatants of
anti-Thy-1 (Y19), anti-CD4 (GK1.5), anti-CD8 (TIB105 and
TIB 210) and anti-Mac1 (TIB 128) antibodies at 4 °C for
30 min. Washed cells were then treated with rabbit
complement (Low-Tox-M, Cedarlane, Accurate Chemical and
Scientific, Westbury, NY, USA) at 37 °C for 30 min. Purified
B cells were washed and resuspended in sterile PBS. The purity
of B cells was greater than 95 % as analyzed by staining with
FITC-conjugated anti-B220 mAb followed by flow cytometry.
Quantification of B cells by flow cytometry
Lymph nodes (LNs) were removed from µMT mice and
gently disrupted between frosted glass slides. Constant
numbers of washed LN cells (0.5×106) were supplemented
with different numbers of purified splenic B cells from wildtype mice. Cell mixtures were stained with FITC-conjugated
anti-B220 mAb and analyzed by flow cytometry as described
above.
Trafficking of B cells in B-cell-deficient mice
Purified B cells were first injected subcutaneously into the
left thigh of µMT mice. After 24 h, single cell suspensions of
lymph nodes and the spleen were analyzed by flow cytometry.
As shown in Fig. 2, the popliteal LN removed from the side of
B-cell injection (left-side cell transfer) was found to contain
the greatest number of B cells (up to 35 % of total cells). Fewer
B cells (5–12 % of the total) were detected in the inguinal LN
(left) isolated from the same side. All lymph nodes examined
on the same side as the cell transfer consistently retained a
greater number of B cells at early times points than
corresponding nodes on the opposite side. Popliteal and
inguinal LNCs opposite to the transfer side contained 7 % or
fewer B cells, while periaortic nodes contained approximately
Results
Detection limits of B cells within lymph node cells in B-celldeficient mice
We first examined the sensitivity of flow cytometry in
detecting B cells within lymph node cells from µMT mice. Half
Popliteal LN
Left
100
Relative cell number
Left
100
35 %
50
Fig. 2. Flow cytometric detection of B cells
subcutaneously transferred into µMT
recipient mice. The plots of fluorescence
intensity histograms illustrate the relative
numbers of B cells stained with anti-B220
monoclonal antibody in single cell
suspensions of different lymph nodes (LN)
and the spleen 24 h after B-cell transfer.
Unstained samples from the same cell
preparation (overlayed heavy lines) were
used to define the area of positive staining
marked by the bars.
Inguinal LN
Right
0
100
101
102
Right
100
7%
50
0
100
101
102
100
11 %
50
0
100
5%
50
0
101
100
102
101
102
Axillary LN
Left
100
100
13 %
50
0
100
0
101
102
100
100
50
100
2%
Spleen
Periaortal LN
Right
50
0
101 102
100 101
Fluorescence intensity
8%
6%
50
0
102
100
101
102
2060 R. ROTH
AND
M. J. MAMULA
8 % B cells after 1 day. Surprisingly, the spleen contained no
more than 6 % B cells after 24 h, indicating that this peripheral
lymphoid organ is not an early reservoir enriched in adoptively
transferred cells. The axillary LN on the injection side
contained approximately four times more B cells than that on
the opposite side. The transient increase in B-cell number in
the popliteal LN on day 3 opposite the subcutaneous transfer
site (see Fig. 3) probably reflects the exit of cells from the
popliteal node nearest to the site of transfer. Background
staining of µMT LNCs or spleen cells with anti-B220 was less
than 1 % of the total staining (Figs 1, 2). All plots in Fig. 2
have an overlay showing the background staining of µMT
LNCs (heavy lines).
We next performed a systematic examination of where B
cells traffic at various times under different routes of adoptive
transfer into µMT mice (Fig. 3). B cell staining is plotted as a
percentage of the number of resident LNCs. As described
above, the greatest number of B cells (35 %) migrated to the
draining popliteal LN nearest to the B-cell injection site within
1 day of subcutaneous injection and remained there for the first
3 days. In contrast, the lowest number of B cells (2.4 %) was
detected in the axillary LN opposite to the B-cell injection side.
One consistent phenomenon of adoptive transfer by the
subcutaneous route was that all lymph nodes (popliteal,
inguinal and axillary) on the side of cell transfer maintained
significantly greater B cell numbers (as much as tenfold
greater) than the corresponding node on the opposite side.
Other routes of adoptive transfer did not display such polarized
trafficking patterns. In contrast to other sites, the periaortal
lymph nodes are only transient reservoirs, with cells detectable
after 24 h leaving again by day 3.
B cells injected intraperitoneally showed a similar
distribution in all LNs and in the spleen. The percentage of B
cells detected ranged between 8.7±0.8 % (inguinal LN, left)
%
20
Popliteal LN
(left)
10
0
Popliteal LN
(right)
10
0
Percentage of stained cells
Periaortal LN
10
0
Inguinal LN
(left)
10
0
Inguinal LN
(right)
10
Axillary LN
(left)
10
0
Fig. 3. Trafficking of B cells in µMT mice. B cells
were injected subcutaneously (A), intraperitoneally
(B) and intravenously (C) into µMT mice. The
lymph node (LN) indicated in the figure and the
spleen were removed 1, 3 and 9 days after B-cell
transfer. Single cell suspensions were stained with
anti-B220 monoclonal antibody and analyzed by
flow cytometry. The percentages of stained B cells
(+1 S.E.M.) among the total LNCs, shown by the
horizontal bars in Fig. 2, are plotted on the y-axes.
Data represent an average of 4–7 experiments.
0
Axillary LN
(right)
10
Spleen
10
0
0
A
A
AA
AA
AA
AA
AA
AA
AA
A
B
C
AA
AA
A
AA
A
A
AA
AA
AA
AA
A AA
AA
AA
AA
A
A
AA
AA
A
Day 1
Day 3
Day 9
B lymphocyte trafficking 2061
Table 1. Absolute numbers of B cells detected in the
lymphoid organs of µMT mice after adoptive transfer
Day 1
Day 3
Day 9
0.89×106
0.59×106
(5.9±1.2 %)
(3.9±1.0 %)
0.25×106
(1.7±0.6 %)
Intraperitoneal
1.2×106
(8.0±0.7 %)
0.73×106
(4.9±0.2 %)
0.36×106
(2.4±0.4 %)
Intraveneous
0.87×106
(5.8±0.3 %)
0.69×106
(4.6±0.9 %)
0.11×106
(0.73±0.3 %)
Subcutaneous
Semi-quantitative analysis of B cells in recipient µMT mice was
determined 1, 3 or 9 days after transfer by the route indicated.
B cell numbers represent the sum of B cells detected in all lymphoid
organs. Total B cell numbers were extrapolated from the signal in flow
cytometry using a standard curve as illustrated in Fig. 1.
Percentages detected among the total number of transferred B cells
(15×106) are expressed in parentheses as mean ± S.E.M., N=5.
and 11.4±1.2 % (inguinal LN, right, and the spleen; means ±
N=4–7) (Fig. 3). A similar trafficking pattern with an
almost equal distribution of the B cells in the LNs and the
spleen, ranging between 6.5 and 10 %, was observed with
intravenously injected B cells 1 day after the transfer.
Three days after the subcutaneous B-cell injection, the
number of B cells detected in the popliteal LN nearest to the
injection side (left) had dropped by 64 % (Fig. 3). A large
decrease (71–87 %) in B cell numbers was also noted in the
periaortal LN of all mice, irrespective of the mode of B-cell
transfer. Also, in all mice, the B-cell number had decreased in
the cell suspensions of the inguinal LN (left) by 20–28 % and
in the axillary LN (left and right) by 37–60 % (Fig. 3). This
loss of B cells was maintained or decreased further by day 9
in all lymph nodes and the spleen. By day 21, B cells were
present in the circulation and numbers began to rise in the
peripheral lymphoid organs, indicating that the transfer
populations were now multiplying after this transient death of
cells (data not shown).
Using the standard curve shown in Fig. 1B, we determined
the absolute number of B cells detected by flow cytometry
(Figs 2, 3) in the lymphoid organs of the µMT recipient mice.
Table 1 shows the total number of B cells (from all harvested
lymph nodes) discovered after subcutaneous, intraperitoneal or
intravenous injection on day 1, 3 and 9. These numbers are also
presented as a percentage of the original number of B cells
injected. Similar B-cell recovery was obtained with all three
types of B-cell transfer, ranging between 5.8 % and 8 % at 1
day post-injection, and between 3.9 % and 4.9 % at 3 days postinjection. Between 0.7 % and 2.4 % of the transferred B cells
were detected 9 days after B-cell injection. Other organs, such
as kidney and liver, were not examined for B-cell populations.
S.E.M.,
Discussion
The present study shows that B-cell trafficking and homing
in the µMT mouse model can be studied by flow cytometry in
a semi-quantitative manner. Prelabeling of the B cells was not
required and thus did not impair the migration or localization
pattern of the transferred cells. Major differences were noted
in the migration pattern of B cells transferred by different
routes after the first day of cell transfer. Subcutaneously
injected B cells localized preferentially to the popliteal LN
nearest the injection site while intraperitoneally or
intravenously injected B cells distributed evenly in the host
lymphoid organs. After 24 h, the distribution of subcutaneously
injected B cells was similar to that of intraperitoneally and
intravenously injected B cells.
Our results contrast with an earlier study in which five times
more B cells (approximately 10 % of the initially injected
radiolabeled cells) were detected in the popliteal LN, while
five times fewer B cells (0.2–0.6 %) were detected in the
pooled mesenteric, inguinal, axillary and brachial LN 2 days
after the subcutaneous transfer of radiolabeled LN B cells into
both hind footpads (Ron and Sprent, 1987). The same authors
also report a tenfold higher B-cell recovery (8 %) in the spleen
and a two- to fourfold lower recovery in the LNs 1 day after
intravenous injection of radiolabeled B cells. It is important
to note that, in the study by Ron and Sprent (1987), mice were
immunized with antigen/CFA 16 h after the B-cell transfer.
Previous studies have demonstrated that antigen/CFA induces
selective recruitment of circulating lymphocytes (Sprent et al.
1971). Thus, antigen treatment may have led to B-cell
accumulation in specific organs because of the inflammatory
responses caused by immunization. It remains to be
investigated whether other differences in the experimental
systems used, such as the source or the labeling of the B cells
or the different B-cell detection techniques applied, may
account for the observed discrepancies. Nevertheless, both
studies demonstrate that intravenously injected B cells
(compared with subcutaneously injected cells) localized in
much lower numbers, 99 % lower (Ron and Sprent, 1987) and
approximately 60 % lower (the present study), in the popliteal
LN at 1–2 days post-injection.
The principal distinction between the present study and
those previously reported is in the phenotype of the recipient
mouse. The present study utilized mice made genetically
deficient in endogenous B lymphocytes. This phenotype in the
recipient mouse makes the identification of transferred cells
accurate and unambiguous. No prior studies of B-lymphocyte
transfer have utilized this unique recipient mouse. However,
we cannot also address the potential effects of cell-to-cell
contact (B cell to B cell) in influencing migration patterns
observed in other studies.
As previously reported by others, large numbers of
transferred B cells (40–80 %) fail to become established in
recipient lymphoid organs (Sprent and Miller, 1972; Ron and
Sprent, 1987). We detected 6–8 % of the initially transferred B
cells in the host lymph nodes and the spleen 1 day after cell
transfer. A slight decrease (1–3 %) was noticed on day 3,
whereas drastically reduced B-cell numbers (0.7–2.4 % of the
initially transferred cells) were detected 9 days after transfer.
The fate of the ‘missing’ B cells is not yet known. Some of the
2062 R. ROTH
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transferred B cells may have died, although it has been reported
that resting B cells survive for at least 2 months when
transferred into recipient mice (Gray, 1988). Space within
secondary lymphoid organs has been suggested to be a major
reason for the more rapid decay of donor LN B cells in nonirradiated recipients as opposed to irradiated hosts (Gray,
1988). However, extreme atrophy of lymphoid tissue appears
not to hinder cell trafficking in nude mice (Sprent and Miller,
1972). It is also unlikely that transferred B cells from syngeneic
donor mice were eliminated by phagocytic cells, such as
macrophages or dendritic cells, although some B cells may
have migrated to other organs such as the liver.
There is no doubt that the µMT mouse model offers a wide
spectrum of biological applications including studies of
mechanistic and functional aspects of B-cell trafficking,
homing and recirculation, or of the fate of B cells in primary
and secondary immune responses. The relevance of the present
work lies in understanding the potential cognate B-cell
interactions that may occur when different routes of adoptive
transfer are used and in predicting the fraction of transferred
cells that may be expected to occur in selected nodes.
We would like to thank Dr Charlie Janeway for his review
of this work and for the contribution of µMT mice. We are also
grateful to Renelle Gee for her technical assistance and to Dr
Mark Shlomchik for the important monoclonal antibody
reagents used in this study. This study was supported by grants
from the Lupus Foundation of America and the NIH (AR41032
and AI36529) to M.J.M.
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