 |
Introduction |
The dynamics of immune responses in vivo are controlled
in part by the migratory patterns of lymphocytes and
APC. Current hypotheses propose that antigen sampling by
dendritic cells (DCs)1 occurs at external surfaces, such as
the skin and mucosal sites, followed by DC migration to
draining lymph nodes, where an immune response is initiated (1, 2). Naive lymphocytes traffic from the blood
stream to lymph nodes via interactions with high endothelial venules, which subsequently allows antigen-specific interactions between naive lymphocytes and DC to occur in
the paracortical regions of the lymph node (3, 4). Following activation, the migratory patterns of lymphocytes are altered such that activated T cells preferentially home to sites
outside of secondary lymphoid tissue. This modification in
trafficking patterns is accomplished via multiple mechanisms, including modulation of homing receptors on lymphocytes as well as modifications in endothelial cell chemokine and counter receptor expression, especially at sites of
inflammation (3, 5).
Lymphocyte homing to the intestinal mucosa has been
extensively studied (8). However, the intestinal mucosal immune system is composed of discrete inductive and
effector sites and, therefore, the requirements for homing
of activated and naive lymphocytes to the anatomically distinct areas may be different. Thus, the inductive sites,
Peyer's patches (PPs) and mesenteric lymph nodes (MLNs),
contain naive as well as activated/memory lymphocytes. In
contrast, the effector sites, lamina propria (LP) and the
intraepithelial lymphocyte (IEL) compartment, contain primarily, if not exclusively, activated or memory lymphocytes (11). The loosely organized tissue of the LP
contains a mixture of CD4 and CD8 T cells, plasma cells,
and memory B cells, whereas IELs are largely CD8+ T cells
expressing either TCR-
/
or TCR-
/
, many of which
express activation and/or memory phenotypes (15). To arrive in the epithelium, IELs presumably transmigrate across
the basement membrane separating the LP and the overlying
epithelium. As the LP lymphocyte and IEL populations are
largely distinct with regard to subsets as well as TCR repertoire, there must exist a highly selective gate between the LP
and IEL compartment, which is likely regulated at the level
of adhesion receptors expressed by subsets of activated lymphocytes and ligands expressed by the basement membrane.
The
4
7 integrin and its ligand, the mucosal addressin
MAdCAM-1 (mucosal addressin cell adhesion molecule 1),
play an important role in entry of activated lymphocytes
into PPs and LP (10). The other member of the
7 integrin
family,
E
7, is highly expressed by IEL (16), but
whether this integrin is involved in migration of cells to the
epithelium or in their retention following entry into the
epithelium is not known (19, 20). Although much has been
learned concerning lymphocyte migration to the mucosa
using in vitro analyses and short-term in vivo adoptive
transfer systems with labeled cells, visualization of trafficking during an ongoing immune response has been made
possible only recently. Moreover, a comprehensive analysis
of the role of integrins in lymphocyte trafficking during an
immune response in vivo has not been performed. The
adoptive transfer of TCR-transgenic T cells (21) or the detection of antigen-specific T cells using MHC tetramer reagents (22) has enabled the monitoring of antigen-specific T cells during immune responses in vivo. Using the
system of transfer of TCR-transgenic T cells followed by
immunization, we have shown that activation is required
for entry of CD8 T cells into the LP and the intestinal epithelium (25, 26). In combination with mice lacking either
all
7 integrins (27) or only the
E
7 integrin, we have
now used this system to analyze trafficking of CD8 T cells
during an immune response in vivo.
 |
Materials and Methods |
Mice.
C57BL/6J (Ly5.1) mice were obtained from The Jackson Laboratory. C57BL/6-Ly5.2 mice were obtained from Charles
River Labs. through the National Cancer Institute animal program.
The OT-I mouse line (28) was maintained as a C57BL/6-Ly5.2
line or on a C57BL/6-RAG-1
/
background (The Jackson Laboratory). OT-I-
7
/
mice and OT-I-
E
/
mice were generated by intercross of the
7
/
(27) or
E
/
mouse lines with
OT-I and screening for
E or
7 and transgenic TCR expression
by flow cytometric analysis of peripheral blood. The
E
/
mouse
line was produced by standard techniques and is described in detail
elsewhere (29).
Recombinant Vesicular Stomatitis Virus Production and Infection.
Vesicular stomatitis virus encoding ovalbumin (VSV-ova) was produced
by ligation of a XhoI-XbaI cDNA fragment containing the entire
ova coding sequence into the pVSV-XN2 vector restricted by XhoI
and NheI (30, 31). The ova gene-containing vector was transfected
along with helper plasmids into BHK cells, and rVSV was recovered
as previously described (30, 31). Ovalbumin production was assessed
by Western blot analysis of detergent lysates and culture supernatants
of infected BHK cells as previously described (26).
Adoptive Transfer.
This method was adopted from Kearney et
al. (21). An equal mixture of 2.5 × 106 pooled LN cells (1.25 × 106
OT-I T cells from OT-I or OT-I-
7
/
or OT-I-
E
/
mice)
were injected intravenously into C57BL/6J (Ly5.1), C57BL/6-Ly5.2, or C57BL/6-Ly5.1/5.2 mice, depending on the donor
cell phenotype. 2 d later, mice were infected with 106 PFU wild-type Indiana serotype VSV (as control) or VSV-ova by intravenous injection. At the later times indicated, cells were isolated
and analyzed for the presence of donor cells by flow cytometric
analysis of Ly5.1/Ly5.2 expression.
Isolation of Lymphocyte Populations.
IEL and LP cells were isolated as described previously (32, 33). Superficial inguinal, axial,
and brachial LNs (peripheral [P]LNs) or MLNs were removed
and single cell suspensions were prepared using a tissue homogenizer. The resulting preparation was filtered through Nitex
(Tetko Industries) and the filtrate centrifuged to pellet the cells.
Immunofluorescence Analysis.
The following mAbs were used
in this study: 53-6.7, anti-CD8
(34); anti-Ly5.1 and anti-Ly5.2
(35); and 2E7, anti-
E integrin (18). mAbs specific for V
2,
V
5, and
7 integrin were obtained from PharMingen as fluorochrome or biotinylated conjugates. For staining, lymphocytes
were resuspended in 0.2% PBS, 0.1% BSA, NaN3 (PBS/BSA/
NaN3) at a concentration of 106-107 cells/ml, followed by incubation at 4°C for 20 min with 100 µl properly diluted mAb. The
mAbs were either directly labeled with FITC, PE, Cy5 (Amersham Life Science), or were biotinylated. For the latter, avidin-
PE-Cy7 (Caltag Labs.) was used as a secondary reagent for detection. After staining, the cells were washed twice with PBS/BSA/
NaN3 and fixed in 3% paraformaldehyde buffer. Four-color analysis of relative fluorescence intensities was then performed with a
FACSCalibur TM (Becton Dickinson). Data were analyzed using LYSYS IITM (Becton Dickinson) or WinMDI software.
 |
Results |
Analysis of
7 Expression on
7- and
E-deficient TCR-transgenic T Cells.
To develop a system for analyzing the
role of
7 integrins in trafficking of naive and activated
CD8 T cells, we introduced TCR transgenes into mouse
lines with disruption of the
7 integrin gene or the
E
integrin gene. T cells of the OT-I mouse line express the
transgene-encoded V
2 and V
5 chains, are predominantly CD8, and recognize the ova peptide, SIINFEKL, in
the context of MHC class I H-2Kb (28). LN cells from
OT-I, OT-I-
E
/
, and OT-I-
7
/
mice were analyzed
for TCR usage and CD8 and
7 integrin expression (Fig. 1).
CD8 cells from the LN of all three strains of mice expressed V
2 and V
5 (representative example shown in
top panel). OT-I CD8 LN cells (either peripheral or mesenteric) expressed heterogenous levels of the
7 integrin,
with a population of
7high cells evident. This population
was absent in OT-I-
E
/
LN cells, indicating that the
high-level expression was due to expression of the
E integrin chain coupled to the
7 chain. This was confirmed by
simultaneous staining for
E and
7 integrins (data not
shown). These patterns of
7 staining are analogous to
what has been observed for nontransgenic naive T cell
populations (36), indicating that despite homogenous TCR
transgene expression, tissue-specific and cell type-specific
heterogeneity in
7 integrin expression remained.

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Fig. 1.
Analysis of 7 integrin expression in OT-I T cells
lacking either 7 or E chains.
Lymphocytes from MLN of
OT-I, OT-I- E / , or OT-I- 7 / mice were examined for
expression of V 2 and V 5 or
for CD8 and 7 by fluorescence
flow cytometry. Similar staining
was observed with PLN cells
(data not shown). Top panel,
analysis of V 2 and V 5 expression of gated CD8+ LN cells
from OT-I mice. Similar results
were obtained from analysis of
CD8+ cells from all three mouse
strains. MFC, mean fluorescence channel.
|
|
Adoptive Transfer System to Determine Requirements for
7 Integrins in Trafficking to Peripheral and Mucosal Inductive Sites.
To allow a direct comparison of migration of normal and
integrin-deficient OT-I cells, we developed a system in
which normal and mutant OT-I cells were mixed in equal
proportions and then transferred to normal mice. By virtue
of Ly5.1/Ly5.2 expression, transferred as well as host populations could be distinguished by flow cytometry (Fig. 2).
In unimmunized mice, OT-I-
7
/
and normal OT-I cells
migrated to PLN (data not shown) and MLN (Fig. 2, top
panel) equally well. 3 d after immunization with rVSV-ova
(26), a substantial increase in OT-I-
7
/
and OT-I cells
had occurred in PLN. This result indicated that
7 integrins were not involved in primary activation of OT-I
CD8 T cells and showed that
7 integrins were not necessary for migration of naive or activated CD8 T cells to
PLN. In contrast, despite similar numbers of normal and
mutant cells in the starting MLN population, 4.5-fold
fewer OT-I-
7
/
cells were present in MLN after infection. All OT-I cells were activated, as determined by an increase in cell size and upregulation of CD44 (reference 26;
data not shown). The population of OT-I-
7
/
cells
present in MLN after infection was likely the result of expansion of those cells present before immunization. Thus,
the difference between this value and the number of control cells may be used as an indicator of the degree of migration into MLN from the peripheral CD8 T cell pool.
When PP lymphocytes were examined after infection, a
barely detectable population of OT-I-
7
/
cells was
found, indicating a near absolute requirement for
7 integrins in migration of activated CD8 T cells to PP.

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Fig. 2.
Tracking in secondary lymphoid organs of cotransferred OT-I and OT-I- 7 /
cells before and after virus infection. Equal numbers of LN cells
from OT-I (Ly5.1+5.2+) and
OT-I- 7 / (Ly5.1+5.2 ) mice
were transferred to normal
C57BL/6-Ly5.2 mice. 48 h
later, mice were infected with
VSV-ova (+VSV) or not (for
naive LN, MLN cells are shown;
PLN cells were similar). 3 d
later, MLN and PLN cells were
isolated and analyzed for the
presence of transferred cells by
flow cytometric detection of
Ly5.1 and Ly5.2 expression.
|
|
Essential Requirement for
7 Integrins in Migration of Naive
Lymphocytes to PP.
The finding that migration to MLN
was apparently different than that to PP (based on a substantial number of OT-I-
7
/
cells in MLN of naive and
immunized mice) suggested that migration of naive OT-I-
7
/
cells to PP may be distinct as compared with MLN.
We tested this possibility by examination of PP 2 d after
transfer of a mixture of naive LN cells from OT-I or
OT-I-
7
/
mice (Fig. 3). This population contains primarily CD8 T cells and B cells. Analysis of total PP CD8 T
cells indicated that
7 integrins were in fact required for
naive OT-I T cells to enter PP, as a distinct population of
normal OT-I but not OT-I-
7
/
cells was detected. Interestingly, examination of transferred B cells also demonstrated a requirement for
7 integrins in their migration to
PP. These results indicated that migration to PP was much
more stringent than migration to MLN, particularly for naive lymphocytes.

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Fig. 3.
7 integrins are required for trafficking of naive
lymphocytes to PP. Equal numbers of LN cells (~60% CD8 T
cells and 40% B cells) from OT-I
(Ly5.1 5.2+) and OT-I- 7 /
(Ly5.1+5.2 ) mice were transferred to normal C57BL/6-Ly5.1+5.2+ mice. 2 d later, cells
from PP were isolated and analyzed for presence of donor populations by detection of CD8,
Ly5.1, and Ly5.2 by flow cytometry. Cells were positively gated
for CD8+ cell analysis and negatively gated (CD8 ) for B cell
analysis. Separate analysis revealed
that the CD8 donor cells were
>95% B cells (data not shown).
|
|
Role of
7 Integrins in Migration of CD8 T Cells to Mucosal
Effector Sites.
The requirement for
7 integrins in homing
to the LP and IEL compartment of small and large intestine
was examined using the adoptive transfer system. After
transfer of naive OT-I cells to normal mice, few if any
transgenic T cells could be detected in LP or IEL (Fig. 4).
After immunization with VSV-ova, a large population of
normal OT-I CD8 cells were present in IEL and LP. However, 7-10-fold fewer OT-I-
7
/
cells were present in
either site, indicating a stringent but not absolute requirement for
7 integrins in migration of activated CD8 T cells
to small intestine effector sites. Similarly, migration of activated CD8 T cells to large intestine IEL was also dependent on
7 integrins (Fig. 4). These results agree well with analysis of
7-deficient mice in which LP and IEL populations
are reduced (27) and suggest that many mucosal effector
cells are derived from cells activated outside of the LP and
IEL compartments.

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Fig. 4.
Migration of activated CD8 T cells to intestinal
mucosa is dependent on 7 integrins. Equal numbers of LN cells
from OT-I (Ly5.1+5.2+) and
OT-I- 7 / (Ly5.1+5.2 ) mice
were transferred to normal
C57BL/6-Ly5.1 5.2+ mice. 48 h
later, mice were infected with
VSV-ova or not. 3 d after infection, mice were killed and lymphocyte populations were isolated and examined for the
presence of donor populations
by detection of Ly5.1/Ly5.2 expression by fluorescence flow cytometry.
|
|
Regulation of
7 Integrin Expression on Antigen-specific T
Cells After In Vivo Immunization.
Because the
7 integrin
requirements for migration to peripheral and mucosal lymphoid tissue were distinct, we analyzed
7 integrin expression of adoptively transferred normal OT-I cells before and
after immunization with VSV-ova. After transfer, naive OT-I cells in PLN and MLN retained low levels of
7 integrin expression (Fig. 5). After immunization of mice with
VSV-ova, OT-I cells in PLN were comprised of two major
populations based on
7 integrin expression. One subset
expressed
7 levels slightly higher than those of naive cells,
and the other population (approximately half the cells) had
high levels of
7 integrins. The majority of this expression
was due to
4
7 expression, as determined by two-color
flow cytometry and analysis of
E
/
OT-I cells (data not
shown). In contrast to activated OT-I cells in PLN, OT-I
cells in MLN and IEL expressed homogeneously high levels of
7 integrins. In these populations, the
E
7 integrin contributed more significantly to overall
7 integrin expression than it did in OT-I cells in PLN (25; data not
shown). These results indicated that activated, but not naive, CD8 cells expressing high levels of
7 integrins preferentially home to MLN and IEL, a finding which correlates
with our demonstrated requirement for
7 integrins in migration of activated CD8 T cells to MLN and PP.

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Fig. 5.
Upregulation of 7
integrins on antigen-specific CD8
T cells after immunization with
VSV-ova. Equal numbers of LN
cells from OT-I (Ly5.1+5.2+) and
OT-I- 7 / (Ly5.1+5.2 ) mice
were transferred to normal
C57BL/6-Ly5.1 5.2+ mice. 2 d
after transfer, mice were infected
with VSV-ova. 3 d later, lymphocytes from the indicated tissues
were analyzed for expression of
CD8, Ly5.1, Ly5.2, and 7 integrin by four-color flow cytometry. Analysis shown is of donor
CD8+ cells. Open histogram,
OT-I- 7 / cells; filled histograms, OT-I cells. Top, naive
MLN cells; naive PLN cells had a
similar expression pattern.
|
|
Analysis of the Role of
E
7 Integrin in Migration of CD8
T Cells During a Primary Immune Response In Vivo.
The data
thus far using
7 integrin-deficient OT-I cells does not allow us to distinguish the relative roles of
4
7 integrin and
E
7 integrin in migration. Although the available literature shows a clear role for
4
7 in migration of lymphocytes to the mucosa, the role of
E
7 in trafficking of lymphocytes remains unclear. Therefore, as described above
for OT-I-
7
/
cells, we transferred a mixture of naive
normal OT-I and OT-I-
E
/
cells to normal mice and
analyzed distribution of naive and activated populations.
Trafficking of naive OT-I cells to PLN, MLN (Fig. 6), or
PP (data not shown) did not require
E
7 expression.
Similarly, after infection with VSV-ova, OT-I and OT-I-
E
/
cells were equally distributed in PLN and MLN
(Fig. 6), indicating that the requirement for
7 integrins in
homing to MLN (Fig. 2) was solely attributable to
4
7.
As with normal naive OT-I cells, naive OT-I-
E
/
cells
did not migrate to LP or IEL (Fig. 7). Moreover, activation via VSV-ova infection resulted in equivalent migration of
OT-I-
E
/
and normal OT-I cells into LP and the IEL
compartment. This result indicated that the
4
7 integrin
was the primary
7 integrin participating in homing of activated CD8 T cells to mucosal effector sites.

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Fig. 6.
Analysis of migration
in LNs of CD8 T cells lacking
E 7. Equal numbers of LN cells
from OT-I (Ly5.1+5.2+) and
OT-I- E / (Ly5.1+5.2 ) mice
were transferred to normal
C57BL/6-Ly5.1 5.2+ mice. 48 h
later, mice were infected with
VSV-ova (+VSV) or not (naive
LN). 3 d later, MLN and PLN
cells were isolated and analyzed
for the presence of transferred
cells by flow cytometric detection
of Ly5.1 and Ly5.2 expression.
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|

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Fig. 7.
E 7 integrin is not required for entry of activated CD8 T
cells into the intestinal mucosa. Equal numbers of LN cells from OT-I
(Ly5.1+5.2+) and OT-I- E / (Ly5.1+5.2 ) mice were transferred to
normal C57BL/6-Ly5.1 5.2+ mice. 48 h later, mice were infected with
VSV-ova or not (naive). 3 d later, IEL and LP cells were isolated and analyzed for the presence of transferred cells by flow cytometric detection of
Ly5.1 and Ly5.2 expression.
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|
Long-Term Retention of CD8 Cells in the Intestinal Epithelium Does Not Require
E
7.
Normal IELs express high levels of
E
7 and low levels of
4
7 (36), supporting the
concept that
E
7 may be involved in tethering of IELs in
the epithelium. We tested whether OT-I cells modulated
their
7 integrin expression following entry into the epithelium by analyzing long-lived mucosal OT-I cells (Fig.
8). 3 wk after immunization, a small population of OT-I cells
was detected in the epithelium (Fig. 8) as well as in peripheral lymphoid organs (data not shown). Analysis of the
long-lived transferred IEL revealed that normal OT-I cells
had high levels of
7 integrins, and this was nearly all attributable to
E
7 expression identical to that of host IEL.
The latter finding was supported by the lack of appreciable
7 integrin expression by long-lived OT-I-
E
/
IEL.
These results indicated that as a consequence of entry into the epithelium,
4
7 was downregulated and
E
7 was
highly upregulated. This phenomenon was not observed
with long-lived nonmucosal OT-I cells (data not shown).
A comparison of the long-term retention of OT-I cells
with or without expression of
E
7 revealed no significant differences between the populations. In the experiment
shown, OT-I-
E
/
IEL were reduced by half as compared with normal OT-I cells (Fig. 8). However, this difference was also evident in PLN and MLN and was not
consistently observed. Thus, at least within the time frame
analyzed,
E
7 and perhaps
4
7 were not required for
retention of CD8 T cells in the intestinal epithelium.

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Fig. 8.
Long-term retention of IEL is not dependent on
E 7 integrin. Equal numbers of
LN cells from OT-I (Ly5.1 5.2+)
and OT-I- E / (Ly5.1+5.2+)
mice were transferred to normal
C57BL/6-Ly5.1+5.2 mice. 48 h
later, mice were infected with
VSV-ova or not (naive). 3 wk
later, IELs were isolated and analyzed for expression of CD8,
Ly5.1, Ly5.2, and 7 integrin by
four-color flow cytometry. Top
panel, 7 integrin expression of
CD8+OT-I- E / IEL (open histogram) and CD8+OT-I- E+/+
IEL (filled histogram).
|
|
 |
Discussion |
The modified adoptive transfer system described here allowed, for the first time, visualization of the role of
7 integrins during an ongoing antiviral immune response in
vivo. By transferring equal numbers of trackable naive normal and
7-deficient antigen-specific CD8 T cells, a direct
comparison of the relative requirement for
7 integrins in
lymphocyte migration before and after in vivo activation
could be made. The system showed clearly that naive CD8
LN cells do not enter the effector sites of the intestinal mucosa, the LP and IEL compartments, whereas naive cells
entered the inductive sites, the MLN and PP. However, an
interesting finding was that the integrin requirements for
trafficking of naive lymphocytes to MLN and PP were distinct. Thus, whereas the
4
7 integrin was not required
for migration of naive LN cells to MLN or PLN, there was
a near absolute requirement for entry of naive CD8 T cells and B cells into PP. The results from our and other studies
(37, 38) indicate that lymphocyte entry into MLN can be
mediated by L-selectin in combination with
4
7 or LFA-1,
and entry into PP requires L-selectin in combination with
4
7 or
4
7 alone, whereas the combination of L-selectin/LFA-1 is not sufficient for lymphocyte entry into PP.
These findings suggest that a functional distinction exists
even within inductive sites of the mucosal immune system,
in that MLN and PP exhibit distinct requirements for lymphocyte entry.
In contrast to the lack of a requirement for
7 integrins
for naive lymphocytes to enter MLN, activated CD8 T cells
relied heavily on this integrin to migrate to MLN. In this
case, our system allowed an estimation of the degree of
CD8 T cell expansion in situ versus the degree of migration to the MLN. That all OT-I-
7
/
cells in MLN after
immunization were activated suggested that these cells
originated from the population of OT-I-
7
/
cells
present in MLN before immunization and underwent in
situ expansion, which is not dependent on
7 integrins. If
in situ expansion was dependent on
7 integrins, then a
significant proportion of nonactivated OT-I-
7
/
cells
should have been detected in the MLN after immunization. There was also a clear early preference for activated
CD8 T cell migration to MLN versus PLN, because after
immunization, the proportion of antigen-specific CD8
T cells was greater in MLN versus PLN. Immunization resulted in a relative ~4-fold increase in OT-I-
7
/
cells in
MLN versus an ~20-fold increase in normal OT-I cells in
the same MLN. If migration accounts for the majority of
this difference, then ~80% of the increase in CD8 T cells
in the MLN was the result of migration rather than in situ
expansion. These results delineated a dichotomy in
7 integrin requirements for entry into MLN, because activated
but not naive CD8 cells required
4
7 for this movement.
The
4
7 integrin was also essential for migration of activated CD8 T cells to PP. This effect was dramatic, as few
naive OT-I-
7
/
cells were present in PP before immunization. In short-term homing assays (90 min), little migration of
7
/
whole spleen cells to PP was detected
(27), in agreement with the results shown here. In the case
of MLN, migration was reduced by approximately half in
the absence of
7
/
(27). As naive
7
/
and
7+/+ CD8
cells homed equally well to MLN in the present system,
these results in short-term migration may indicate supplantation of
7 by other molecules in the longer term (37) or
could be due to decreased binding of non-CD8+ cells or
poor binding of memory lymphocytes contained in the spleen populations. Nevertheless, these results indicated a
near absolute requirement for
4
7 to direct migration of
naive and activated lymphocytes to PP, whereas the requirements for entry of naive and activated cells to MLN
were different.
Although migration of CD8 T cells to PP was totally dependent on
7 integrins, some
7-independent migration
of activated CD8 T cells to intestinal mucosal effector sites
was noted. Naive OT-I cells did not enter the LP or IEL
compartments, whether they expressed
7 integrins or not.
After immunization, however, large numbers of activated
CD8 T cells entered the LP and the epithelium. This migration begins ~48 h after immunization (25, 26; data not shown). Only ~85-90% of this migration was
7 integrin
dependent, as determined by comparison of migration of
normal and
7
/
OT-I cells. This was true in small intestinal LP and the IEL compartments of small and large intestine. This was a conservative estimate, as the adoptive
transfer system may overestimate the significance of
7 integrins due to competition between normal and mutant
cells. That migration to the IEL compartment required, for
the most part,
7 integrins suggested that either
4
7 interaction with basement membrane allows CD8 cells to enter the epithelium from the LP or the inhibition of entry to
the LP resulted in decreased availability of cells for entry
into the IEL population. Further analysis of other adhesion
molecules will be necessary to answer this question. The
demonstration of some
7-independent migration indicated that other adhesion molecules are capable of directing migration to the LP and the epithelium. Although
4
7 is
thought to be the major participant in contact, rolling,
arrest, and diapedesis of lymphocytes homing to LP (3), we
previously demonstrated that
2 integrins and intracellular
adhesion molecule 1 are important for the establishment of
the CD8
LP and IEL populations (39). These findings
suggest that the
2 integrins, perhaps LFA-1, may be able
to direct some level of homing to intestinal effector sites.
Perhaps other adhesion molecules, such as L-selectin, are also involved in
7-independent homing to mucosa.
In contrast to the well-described functions of the
4
7
integrin, the function of the
E
7 integrin in vivo remains
unknown. The only known ligand for
E
7 is E-cadherin,
which is highly expressed by intestinal and other epithelial
cells (19, 40). Because IEL half-life is much less than that of
epithelial cells, this finding supported the hypothesis that
IEL are retained in the epithelium via an
E
7-E-cadherin
interaction. As
4 integrin-deficient mice have normal IEL
numbers (41), whereas
7 integrin-deficient mice have reduced IEL numbers (27), these results also support the possibility that
E
7 is involved in some aspect of IEL migration or retention. However, the data shown here do not
support this concept. The absence of the
E integrin chain
on OT-I cells had no effect on their ability to migrate to
the LP or the epithelium after primary activation, indicating that all
7-mediated migration was
4
7 dependent.
Moreover, OT-I cells with or without
E expression were
retained equally well in the epithelium for up to 3 wk. Indeed, the long-lived cells expressed low levels of
7 integrins, suggesting that
4
7 was also not involved in long-term retention of IEL in the epithelium. Therefore, other
adhesive molecules must be involved in allowing long-lived IEL to be maintained in the epithelium, perhaps via
interactions with the basement membrane rather than the
epithelial cells. Given the in vitro data, it seems likely that
the
E
7 integrin plays an adhesive role not measured by our system. What remains unknown is the resulting functional outcome of such an interaction.
Address correspondence to Leo Lefrançois, UCONN Health Center, MC1310, Department of Medicine,
263 Farmington Ave., Farmington, CT 06030. Phone: 860-679-3242; Fax: 860-679-1287; E-mail: llefranc{at}panda.uchc.edu
This work was supported by National Institutes of Health grants AI41576 and DK45260 and an American
Cancer Society Faculty Research Award to L. Lefrançois and by a grant from the Deutsche Forschungsgemeinschaft (WA 1127/1-1) to W. Muller and N. Wagner.
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