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T Cell Subsets Express Distinct Patterns of Chemokine Responsiveness and Adhesion Molecules: A Mechanism for Tissue-Specific T Cell Subset Accumulation1
* Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, and Center for Molecular Biology and Medicine, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304;
Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717; and
Millennium Pharmaceuticals, Cambridge, MA 02139
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells localize to distinct tissue sites
in the absence of exogenous Ag stimulation or development of
effector/memory cells. Selective lymphocyte homing from the blood into
tissues is controlled by a multistep process involving vascular and
lymphocyte adhesion molecules, and G protein-linked chemokine
receptors. The role of these mechanisms in the tissue tropism of
T cells is still poorly understood. In this study, we demonstrate that
a subset of T cells, most of which express an antigenically
distinct TCR and are characterized by coexpression of CD8, selectively
accumulated in tissues that expressed high levels of the mucosal
vascular addressin, mucosal addressin cell adhesion molecule 1.
These cells expressed higher levels of 
4
7
integrins than other T cell subsets and selectively migrated to
the CCR7 ligand secondary lymphoid-tissue chemokine (CCL21).
Integrin activation by CCL21 selectively increased
CD8+ T cell binding to recombinant mucosal addressin
cell adhesion molecule 1. These results suggest that the tropism of
circulating CD8+ T cells for mucosal tissues is due,
at least in part, to selective developmental expression of adhesion
molecules and chemokine receptors. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells; however, many
aspects of this population are still poorly understood. One intriguing
characteristic demonstrated in studies with mice, cattle, nonhuman
primates, and humans is that discrete subsets of T cells,
defined by their TCR usage, specifically accumulate in certain tissues
and organs in the absence of exogenous Ag stimulation (reviewed in Ref.
1). For example, mouse T cells associated with the
epidermis (2, 3) and the mucosal epithelia of the vagina,
uterus, and tongue (4, 5) express distinct combinations of
- and -chains. Other distinct TCR-defined subsets of T
cells are found in the blood, spleen, intestine, lung, liver, and
mammary glands (reviewed in Ref. 1). The homing and
accumulation of T cells is of particular interest, as the
presence or absence of TCR-defined subsets of T cells
dramatically influences the outcome of some infectious diseases
(6, 7). Thus, defining the molecular events that control
the selective tissue distribution of these cells is warranted.
Cattle provide a useful model for the study of T cells because,
unlike in adult humans, these cells constitute a predominant population
of lymphocytes in young animals. One tissue-specific T cell
subset in cattle expresses CD8 and CD2, lacks the GD3.5 Ag, and most
cells within the population express an antigenically distinct TCR
(8, 9, 10) (referred to in this study as
CD8+ T cells). Bovine
CD8+ T cells are found in increased numbers
in the spleen, gut lamina propria, and mesenteric lymph nodes
(MLN).4 Conversely,
other TCR-defined subsets of T cells (which do not express CD8)
are found predominantly in blood, peripheral lymph nodes (PLN),
and skin (9, 10). The ability to collect large numbers of
peripheral blood T cells, as is possible in calves, provides a
unique opportunity to study the molecular mechanisms controlling the
tissue-specific accumulation of these cells.
Leukocytes are generally recruited from the circulation into tissues
through selective interactions with the vascular endothelium. A
multistep process mediated through a series of adhesion and signaling
molecules present on both lymphocyte and endothelial surfaces is
responsible for the slowing, arrest, and transendothelial migration of
circulating leukocytes (11). Adhesion molecules
(selectins, integrins, and their corresponding ligands) participate in
tethering, rolling, and adhesion of lymphocytes. Chemokines can
function as critical regulators of lymphocyte homing by triggering
integrin-dependent arrest of rolling lymphocytes, as well as subsequent
transendothelial migration into tissues (12, 13). Analysis
of the role of chemokines in T cell subset homing and
accumulation has been limited, due, in part, to the small numbers of
T cells in the mouse, making functional assays difficult.
In our initial study of primary bovine T cells, we found that
most CD8+ T cells, unlike
CD8- T cells, do not express L-selectin
and E-selectin ligands and do not efficiently home to sites of s.c.
inflammation (10). The molecular mechanisms that
contribute to the selective homing and accumulation of
CD8+ T cells into mucosal tissues have not
been determined. In the current study, we show that the majority of
peripheral blood CD8+ T cells express
functionally higher levels of the
47 integrin than
CD8- T cells. Additionally,
CD8+ T cells selectively migrate to the
CCR7 ligands secondary lymphoid-tissue chemokine (CCR ligand (CCL)21)
and macrophage inflammatory protein-3 (CCL19) in in
vitro chemotaxis assays. CCL21 stimulation also results in
significantly increased efficiency of CD8+
T cell binding to mucosal addressin cell adhesion molecule 1
(MAdCAM-1), compared with CD8- T cells.
These results demonstrate that functional differences in adhesion
molecule expression and chemokine sensitivity in T cell subsets
likely contribute to their unique accumulation patterns in
vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Holstein calves were purchased from local producers and housed at the Montana State University Large Animal Facilities at the Veterinary Molecular Biology Laboratory (Bozeman, MT). Cattle used in this study were bull calves from 1 to 6 mo of age. Tissue samples were collected from animals upon necropsy and were cut and frozen in OCT freezing medium (Tissue-Tek, Elkhart, IN). For immunohistology, frozen sections were air-dried, fixed in acetone, and stained as previously described (14).
Cells and cell lines
Peripheral blood was collected into sodium heparin anticoagulant tubes by venipuncture and PBMCs were purified by Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO) gradient centrifugation. Lymphocytes were isolated from the thymus, lymph nodes, and spleen by first finely mincing tissues with a razor blade. Samples were then dounced in a tissue homogenizer to release lymphocytes from the stromal elements. Cell suspensions isolated from various tissues were then passed through a layer of Nytex fabric (Fairmont Fabrics, Hercules, CA) to remove cell aggregates, and the resulting cell preparation was stained for FACS analysis or used in functional assays. Intraepithelial lymphocytes were isolated by exteriorizing the lumen of the small intestine, washing extensively with PBS, and gently scraping the luminal surface with a razor blade. Cell scrapings were then dounced in a tissue homogenizer, and isolated as described above.
Mouse MAdCAM-1 transfected Chinese hamster ovary (CHO) cells and nontransfected CHO cells have been previously described (15).
mAbs used in this study
The following mouse mAbs were used: GD3.8, which recognizes a
pan epitope on the bovine TCR (8); GD3.5, which
recognizes a lineage-specific Ag on T cells (16);
CC58, which recognizes bovine CD8, kindly provided by C. Howard
(Institute for Animal Health, Compton, U.K.) (17); MHM23,
which recognizes human and bovine CD18 (DAKO, Carpinteria, CA);
FW4-101, which recognizes bovine CD29 (VMRD, Pullman, WA); HP2/1, which
recognizes CD49d (Serotec, Raleigh, NC); and 7G11, which recognizes
human MAdCAM-1 and cross-reacts with ovine and bovine MAdCAM-1
(18). The following rat mAbs were used: FIB30, a rat
anti-7 integrin mAb that has previously
been shown to specifically bind human and mouse
7 integrin (19) and cross-reacts
with bovine 7; and MECA367, a
function-blocking mAb which recognizes mouse MAdCAM-1
(20). SK208, a rat mAb which recognizes mouse neutrophils
(M. A. Jutila, unpublished observations), and EL81
(21), a mouse mAb that recognizes human E-selectin, were
used as isotype-matched negative control mAbs. PE-conjugated
anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove,
PA), and avidin-conjugated CyChrome (BD PharMingen, San Diego, CA)
were used as secondary reagents.
Three-color FACS analysis and cell sorting
FACS analysis was performed as follows: 100 µl of hybridoma supernatant fluid or 100 µl of purified mAb at a final concentration of 50 µg/ml was incubated with cells for 30 min on ice and then washed from the cells with PBS containing 5% horse serum (PBS-HS; Sigma-Aldrich). PE-labeled second stage diluted 1/250 in PBS-HS was then added and incubated for 30 min on ice. The samples were washed with PBS-HS, incubated in 10% mouse serum in PBS for 15 min, and washed again. Biotin-labeled GD3.8 and FITC-conjugated CC58 were then added and the cells were incubated on ice for 30 min. Cells were then washed in PBS-HS and incubated with avidin-CyChrome diluted 1/2000 in PBS-HS. After 30 min on ice, the cells were washed in PBS-HS and analyzed using a FACSCalibur (BD Immunocytometry Systems, Mountain View, CA). FL1 (FITC), FL2 (PE), and FL3 (CyChrome) detectors were used and the FACSCalibur was calibrated using Calibright beads (BD Immunocytometry Systems). Compensation was set manually using single-color stains of the various fluorochromes. Data from up to 100,000 cells was acquired and reported in two-dimensional dot plots or histograms. Negative controls included 1) single-color stains, 2) irrelevant isotype-matched Ab stains, and 3) second-stage reagent controls. For statistical analysis, markers were placed just above the upper limit of background staining of control Abs. We found no evidence that the CyChrome reagent bound nonspecifically, as previously described for some reagents conjugated to this fluorochrome (22, 23).
Different sorting approaches were tested for generating highly pure
populations of CD8+ and
CD8- T cells for detailed real-time
quantitative RT-PCR described in mRNA analysis. Sorts
using CC58 or another marker (GD3.5 mAb; Refs. 10 and
16) in combination with GD3.8 were compared). The
GD3.5 mAb distinguishes the inflammatory CD8-
from the CD8+ T cell. Specifically, GD3.5
stains CD2-CD8- T cells in
neonates (10). Greater than 90–95% of the
GD3.5- cells are CD8+ and
virtually 100% of these cells express CD2 (10, 16).
Stained T cell subset populations were sorted at 10,000 cells/s
on a FACSVantage cell sorter (BD Immunocytometry Systems). Purity of
the sorts was confirmed by analysis of the sorted populations on a
FACSCalibur.
Chemotaxis assays
Bovine PBMC were analyzed in chemotaxis assays using transwell
24-well tissue culture inserts (5-µm pore size; Corning, Corning, NY)
as previously described (24). Briefly, 1 x
106 cells were added to the insert in 100 µl of
medium. Chemokine preparations, or medium alone, were added to the
lower well. All migration assays were done in RPMI 1640 with 10%
bovine serum at 37°C plus 10% CO2 for 2
h. Optimal concentrations of chemokines (all from PeproTech, Rocky
Hill, NJ) were determined and the following concentrations were used in
each experiment: 10–15 nM recombinant human stromal cell-derived
factor-1 (CXC ligand (CXCL)12); 100 nM recombinant murine
CCL21; and 150 nM recombinant murine CCL19. Phenotypic analysis and
quantification of the migration of the T cell subsets was
determined by a flow cytometric approach, as previously described
(24). Briefly, following migration, 50,000 15-µm
polystyrene beads were added to each sample as an internal counting
standard. The ratio of beads to each lymphocyte subset was determined
for the total input cells, and the cells that migrated into the lower
chambers, and was used to calculate the percentage of migration of each
cell subset. T cell subsets were determined by multicolor FACS
analysis, as described above.
MAdCAM-1 binding assay
Bovine peripheral blood was collected and PBMC-purified, as described in Cells and cell lines. Mononuclear cells were then incubated for 24 h at 37°C in T-175 flasks (Nunc, Naperville, IL) containing RPMI medium supplemented with 10% FBS. The nonadherent lymphocyte population was washed in HBSS (Life Technologies, Grand Island, NY) and resuspended at 5 x 106 cells/ml in DMEM/BSA before use in functional assays. Adhesion assays were performed in T-25 flasks containing confluent monolayers of MAdCAM-1-transfected or nontransfected CHO cells. Briefly, MAdCAM-1 binding cells were separated from whole lymphocyte preparations by incubating 1 x 107 PBMC in 2 ml of medium under constant rotation on a horizontal rotator (30 rotations/min) for 10 min. Unbound cells were decanted and the flasks were washed twice in HBSS. Lymphocytes bound to CHO cells were then eluted by treatment with 2 mM EDTA/PBS. Following elution of adherent cells, polystyrene beads were added to each sample to facilitate counting. The phenotype and number of cells collected from adhesion assays was then determined by multicolor flow cytometric analysis, as described above. In experiments to test the effects of chemokines on lymphocyte adhesion, 100 nM soluble CCL21 or 15 nM CXCL12 (final concentration) was added for the final 2 min of the 10-min incubation. Specificity controls for MAdCAM-1 mediated adhesion of lymphocytes included the MAdCAM-1 blocking mAb MECA367 (50 µg/ml) and the isotype-matched negative control mAb SK208 (50 µg/ml).
mRNA analysis
Bovine PBL were collected by histopaque gradient centrifugation
at 1300 x g, and sorted into
CD8+ and CD8- T cell
populations. Preliminary analysis suggested that mRNA degradation
occurred immediately following the sorting procedure (data not shown).
Therefore, sorted cells were rested overnight in complete RPMI medium,
then treated for 4 h with 5 µg/ml Con A and 1 ng/ml recombinant
human IL-2 to stimulate transcription of message down-regulated during
the 
8-h staining and sorting process. RNA was extracted from 1
x 107 cells (pooled from multiple animals) of
each population with TRIzol (Life Technologies) according to the
manufacturer’s instructions. The RNA was treated with DNase for 1
h at 37°C and extracted again with phenol-chloroform (1:1),
and precipitated with a 0.1 volume of 5 M NaCl and a 2.5 volume of
ethanol. Total bovine PBLs were similarly stimulated and the RNA was
extracted for use as template to construct standard curves.
Relative CCR7-specific RNA message in CD8+
and CD8- T cells was quantified by
measuring SYBR green incorporation during real-time quantitative RT-PCR
using the relative standard curve method (without determining the exact
quantity of target RNA). Bovine expressed sequence tags
homologous to human and mouse CCR7 sequences were selected. A contig of
bovine CCR7 was then constructed from the sequences with accession
numbers AW347844, AW447807, AW632094, and BF073678 and the Vector NTI
software (Applied Biosystems, Foster City, CA). Bovine
CCR7-specific primers were designed using the Primer Express software,
and the pair with the lowest penalty was selected (5' primer:
5'-AGCACGTGGAGGCCTTGAT-3', 3' primer:
5'-GCGGATGATGACGAGGTAGC-3'). Primers specific for bovine 18S
RNA (endogenous control) and 7 integrin were
similarly designed (7 5' primer:
5'-CCTGCTGGTGTTCACGTCAG-3', 7 3' primer:
5'-GCTGCGACTGTAGAGGCCAT-3'. 18S 5' primer:
5'-TCGAGGCCCTGTAATTGGAA-3', 18S 3' primer:
5'-CCCAAGATCCAACTACGAGCTT-3'). The standard curve was constructed using
total bovine PBL RNA starting at 4 µl per 20 µl reverse
transcription (RT) reaction, diluted serially to 0.25 µl per
RT reaction. The RT was performed with Superscript RT and random
primers (Life Technologies) according to the manufacturer’s protocol.
One microliter of each RT reaction was subsequently used in the PCR.
The PCRs used to generate the standard curve were performed in
duplicate, and the T cell subset samples were performed in
triplicate. The PCR was cycled and data was collected on the Applied
Biosystems GeneAmp 5700 sequence detection system (Foster City, CA) and
all calculations were performed as described in user bulletin no. 2 for
the ABI PRISM 7700 sequence detection system.
Statistical analysis
Results were analyzed using the paired Student t test. Significant p values are indicated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The distribution of bovine MAdCAM-1 closely resembled the MAdCAM-1
distribution described in the mouse (20, 25), with
high expression at mucosal sites and minimal expression in peripheral
tissues. MAdCAM-1 was expressed at high levels on venules in the MLN as
well as throughout the ileal mucosa and the lamina propria (Fig. 1
). Most PLN examined had undetectable
levels of MAdCAM-1; however, in some PLNs, minimal expression of
MAdCAM-1 was occasionally seen in the paracortical area. This
observation is similar to the PLN expression of MAdCAM-1 described in
sheep (18). A considerable amount of MAdCAM-1 was
expressed in the splenic marginal zone, extending into the red pulp
area (Fig. 1). MAdCAM-1 expression was not seen in the skin or the
thymus (data not shown).
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T cells accumulate in tissues expressing
MAdCAM-1
In an effort to determine whether subsets of T cells
accumulated in sites expressing MAdCAM-1, T cell subset analysis
of various mucosal and nonmucosal organs was performed. The percentage
of T cells as a function of total lymphocytes varied greatly
from tissue to tissue as previously reported (26, 27, 28, 29).
However, the percentage of CD8+ T cells as
a function of total T cells correlated directly with the amount
of MAdCAM-1 expressed in the tissue (Table I). For example, at sites expressing high
levels of MAdCAM-1, such as the intestinal lamina propria and the MLN,
large numbers of T cells coexpressed CD8. In contrast, in PLN,
which contained minimal levels of MAdCAM-1, the percentage of
CD8+ T cells was similar to that found in
peripheral blood (Table I).
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T cells express high
levels of 4 and 7 integrins
To determine whether CD8+ T cells in
the circulation express the MAdCAM-1 counter receptor
47, FACS analysis of
T cell subsets was performed. Multicolor FACS analysis of bovine
peripheral blood T cells showed that the fluorescence of the
anti-7 integrin stain on
CD8+ T cells (mean 554) was 1.5 fold
higher (p < 0.01) than that on
CD8- T cells (mean 322) (Fig. 2). The expression of the
4 integrin on CD8+
T cells was also higher than on CD8- T
cells, but expression of the 1 (CD29) and
2 (CD18) integrins did not differ
statistically between the two populations (data not shown).
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T cells preferentially bind
recombinant mouse MAdCAM-1
To determine whether the different levels of integrin expression
on CD8+ and
CD8- T cells correlated with functional
differences in adhesion, we examined the ability of these subsets
to bind MAdCAM-1. As expected, a variety of cells in the PBL
preparations bound MAdCAM-1, including T cells (data not shown),
B cells (data not shown), and T cells (Fig. 3). CD8+ T
cells were recovered at a higher frequency (2.5-fold) than
CD8- T cells (p <
0.01) (Fig. 3), indicating that the differences observed in
4 and 7 expression
levels by flow cytometry, resulted in measurable functional differences
in the ability of these cells to bind MAdCAM-1. However, the overall
level of binding of the untreated cells was low, suggesting that only a
fraction of the total cells in either population expressed activated
47 integrins.
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, T cells
migrate to soluble CCR7 ligands
Transwell migration chambers were used to analyze the chemotactic
response of bovine T cell subsets to various mouse and human
chemokines. Recombinant mouse CCL21 and CCL19 and human CXCL12 were
shown to cross-react in the bovine system and mediate the migration of
bovine lymphocytes (data not shown and Fig. 4). CXCL12 elicited an equally robust
response by both T cell subsets (Fig. 4). In contrast, CCL21
(Fig. 4) and CCL19 (data not shown), which bind the CCR7 chemokine
receptor, preferentially stimulated the directional migration of
CD8+ T cells.
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T cells to MAdCAM-1-transfected CHO cells
Chemokine-mediated activation of
47 has been shown to
result in the firm adhesion of rolling lymphocytes to MAdCAM-1
(30, 31, 32). Therefore, we next tested whether the minimal
47/MAdCAM-1 binding
shown in Fig. 3
could be augmented by chemokine activation in a
T cell subset-specific fashion. The same MAdCAM-1 adhesion assay, with
the addition of chemokine for the final 2 min of the 10-min adhesion
assay, was used. The addition of soluble CXCL12 resulted in an increase
of T cell binding to MAdCAM-1, with both T cell subsets
increasing 4-fold over the levels recovered from MAdCAM-1 binding
assays without CXCL12. In contrast, binding assays in which CCL21 was
added resulted in a selective 4-fold increase in the binding of
CD8+ T cells with no increase in the
binding of CD8- T cells (Fig. 5).
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7 integrin in
sorted CD8+ and CD8- T cells
Sorting approaches were established to compare CCR7 and
7 transcript levels in
CD8+ and CD8- T
cells by real-time quantitative RT-PCR. Initial sorts using
anti-CD8 mAb were ineffective (purity of the
CD8+ cells preparation averaged 60%, data not
shown). As such, GD3.5 mAb, which recognizes inflammatory
CD8- T cells (with >90–95% of the
GD3.5- T cells being
CD8+; Refs. 10 and 16),
was used. Use of GD3.5 mAb plus GD3.8 provided sorts that were >98%
pure (data not shown), which were used in real-time RT-PCR analysis of
CCR7 and 7 transcripts. The results of the
CCR7 and 7 standard curves can be summarized
by the slope, y-intercept, and
R2 values of the resulting lines
(y = -5.2486x + 26.373,
R2 = 0.938 and y =
-5.4098x + 13.587, R2 = 0.9502,
respectively). These values were used to calculate the volume of target
RNA in the two T cell subsets. The values were then normalized
to 18S by division with the corresponding 18S value and the SD
determined. The results indicated that the
GD3.5- T cells contained significantly
greater amounts of CCR7 transcripts than GD3.5+
T cells (>3-fold), even though both populations were sorted and
cultured under identical conditions. As another comparison, levels of
7 transcripts were also analyzed in both
subsets. GD3.5- T cells had significantly
greater amounts of 7 transcripts than GD3.5+
T cells (Fig. 6). Because the
predominant GD3.5- T cell population is
CD8+ and virtually all
GD3.5+ T cells are
CD8- in the animals used in this study, we
conclude that the differences in transcript levels were due to
differences in the CD8+ and
CD8- subsets, which is consistent with the flow
cytometric analysis reported above.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells. Although tethering and rolling of
leukocytes on the endothelium is an essential first step in the
migration process, ultimately, the migration of a cell requires
chemokine-mediated integrin activation, leading to tight adhesion and
finally migration along a chemotactic gradient. The chemokine CCL21 is
of particular interest in that CCL21/CCR7 interactions have been shown
to play a pivotal role in the migration of T cells into secondary
lymphoid organs (33, 34, 35). Furthermore, CCR7 ligands have
been shown to induce rapid firm adhesion of lymphoid cells rolling on
MAdCAM-1 in vitro and in vivo (30, 31). Our data
demonstrate that CCL21 selectively mediates chemotaxis and augments
MAdCAM-1 binding of CD8+ T cells which
provides a mechanism to explain the preference of this cell population
for MAdCAM-1-expressing secondary lymphoid tissues. Indeed, increased
frequency of CD8+ T cells correlates
directly with high levels of MAdCAM-1 in Peyer’s patch and MLN.
Although some CD8- T cells express high
levels of 47 and
small numbers specifically bind to MAdCAM-1 transfectants, the addition
of CCL21 to binding assays does not increase the number of
CD8- T cells binding MAdCAM-1. Thus, as a
population in circulation and in the absence of active immunization/Ag
challenge, CD8+ T cells express the
necessary chemokine receptors and adhesion molecules that likely direct
their homing to MAdCAM-1-expressing secondary lymphoid tissues. The
finding that CD8+ T cells are found in
increased numbers in the lamina propria, a site which has not been
shown to express CCL21, suggests that other chemokines may play a role
in the homing of these cells to some tissues. The chemokines
thymus-expressed chemokine (CCL25) and mucosae-associated epithelial
chemokine (CCL28) which have been shown to be expressed within the
small and large intestines (36, 37, 38) or more globally
expressed chemokines, such as CXCL12, may play a role in
CD8+ T cell homing to nonsecondary lymphoid
mucosal tissues.
Analysis of RNA levels shows that the differences in integrin and
chemokine receptor expression in CD8+ and
CD8- T cells (as defined by the GD3.5 mAb)
are likely controlled at the level of gene transcription. These
results, in addition to our earlier studies (10), suggest
that the differentiation process leading to the segregation of the
CD8+ and CD8- T cell
populations, leads to dramatic gene regulation events that alter the
function of both populations. An ongoing functional genomic analysis of
each subset supports this view and suggests that over 300 genes are
differentially regulated in these subsets. The genes expressed by
CD8- T cells are consistent with an
inflammatory or activated phenotype, whereas, many of the genes
expressed by CD8+ T cells are
anti-inflammatory (N. Meissner, J. Hedges, M. A. Jutila,
unpublished observations). The selective chemokine receptor and
adhesion molecule profiles of these subsets likely ensures that the
subset with the appropriate functional activity is delivered to the
"right" tissue. The factors that regulate the gene expression
patterns leading to the trafficking phenotypes of the
CD8+ and CD8- T
cells are currently under investigation.
Although no differential adhesion molecule or chemokine receptor
expression has been previously described for human T cell
subsets that account for tissue-specific accumulation, Glatzel et al.
(39) have recently shown that the chemokine receptors CCR5
and CXCR1 are differentially expressed on V2 and V1 cells. In
their study, the authors showed that CCR5 is expressed predominantly on
CD45RO V2V9 cells and suggested that these cells represent a
population of phospho-Ag-activated effector/memory cells, rather than
naive T cells. Although CXCR1 is expressed predominantly on
V1 cells (39), a previous study, which analyzed total
T cell migration, did not show a response to the CXCR1 ligand
IL-8 (40). However, V1 cells typically constitute a
small minority of T cells in the peripheral circulation, thus
additional work is needed to determine whether CXCR1 mediates specific
migration of these cells. In preliminary experiments, we analyzed the
migration of circulating adult human CD8- and
CD8+ T cells in response to CCL21 and did
not detect differences in the migration of the two subsets. Perhaps
just as importantly, differences in
47 expression were
likewise not detected. Additionally, there are differences in the type
of CD8 molecule found on T cells from adult humans vs those in
newborn ruminants, with respect to the type of CD8 molecule they
express, which likely impacts these results. In humans, many
CD8+ T cells express the CD8
homodimer (41); whereas, most circulating
CD8+ T cells in young cattle express the
CD8 heterodimer (42). Thus, if a human correlate to
the results reported in this study exists, it may reside in a subset of
the overall CD8+ pool of T cells (those
that express the CD8 heterodimer, for example) or at different
stages of development (i.e., in neonates). In support of this
possibility, we have found that in human T cells expanded in
vitro by the addition of phospho-Ag preparations from
Mycobacterium sp, plus IL-2/IL-15, the
CD8+ percentage increases and these cells express
higher levels of 7 than
CD8- T cells (J. F. Hedges and
M. A. Jutila, unpublished observations). It may be that human
CD8+ T cells, which selectively respond to
certain infectious agents and/or are defined by specific V and/or
V usage, are phenotypically similar to
CD8+ T cell in calves.
In conclusion, through the use of functional analyses of primary cells,
we have demonstrated that T cell subsets exhibit functional
differences in respect to both the adhesion molecules expressed on
their cell surface and their responsiveness to chemokines. The
functional differences described in this study show clear differences
in subsets that may result in differential regulation of these
cells at each step of the multistep model of lymphocyte homing.
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Acknowledgments |
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Footnotes |
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2 Current address: Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine and Center for Molecular Biology and Medicine, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304.
3 Address correspondence and reprint requests to Dr. Mark A. Jutila, Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717. E-mail address: uvsmj{at}montana.edu
4 Abbreviations used in this paper: MLN, mesenteric lymph node; PLN, peripheral lymph node; CCL, CCR ligand; MAdCAM-1, mucosal addressin cell adhesion molecule 1; CHO, Chinese hamster ovary; PBS-HS, CXCL, CXC ligand; RT, reverse transcription.
Received for publication July 25, 2002. Accepted for publication August 28, 2002.
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J. F. Hedges, J. C. Graff, and M. A. Jutila |
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