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IMMUNOBIOLOGY
Function of CD4⫹CD3⫺ cells in relation to B- and T-zone stroma in spleen
Mi-Yeon Kim,1 Fiona M. McConnell,1 Fabrina M. C. Gaspal,1 Andrea White,1 Stephanie H. Glanville,1 Vasilios Bekiaris,1
Lucy S. K. Walker,1 Jorge Caamano,1 Eric Jenkinson,1 Graham Anderson,1 and Peter J. L. Lane1
1Medical
Research Council (MRC) Centre for Immune Regulation, Institute for Biomedical Research, Birmingham Medical School, Birmingham, United Kingdom
Lymphocytes from lymphotoxin (LT)
␣–deficient mice, which lack segregation
of their B- and T-cell areas, acquire normal organization following adoptive transfer into RAG-deficient recipients, identifying a non-B non-T cell in the segregation
process. Here we show that a CD4ⴙCD3ⴚ
accessory cell is tightly associated with
discrete VCAM-1–expressing stromal
cells in B- and T-cell areas of the mouse
spleen. CD4ⴙCD3ⴚ cells express high levels of LT␣, LT␤, and tumor necrosis factor
(TNF) ␣, which are the ligands for the LT␤
receptor and TNFR1 expressed by stromal cells. The expression of these ligands is functional, as transferring
CD4ⴙCD3ⴚ cells derived from either embryonic or adult tissues into LT␣deficient mice organizes B/T segregation
and up-regulates CCL21 protein expres-
sion in areas where T cells are segregated from B cells. We propose that the
function of CD4ⴙCD3ⴚ cells is to form a
link between primed CD4 T cells and the
underlying stromal elements, creating distinct microenvironments in which they
enable effector responses. (Blood. 2007;
109:1602-1610)
© 2007 by The American Society of Hematology
Introduction
The development of segregated B-cell and T-cell areas within
secondary lymphoid organs is the platform for the development of
both high-affinity class-switched antibodies and memory antibody
responses; neither of these functions develops in lymphotoxin (LT)
␣⫺/⫺ mice, in which there is no B/T segregation.1 The absence of
segregation is due to impaired organization rather than intrinsic
defects in the lymphocytes themselves, as LT␣⫺/⫺ lymphocytes
both segregate and function normally following transfer into
irradiated normal2 or RAG-deficient1 hosts, which lack B and
T cells. A cellular source other than mature B or T cells is therefore
implicated in the process of organization.
LT␤R signals and perhaps TNFR1 signals mediate lymphoid
B/T segregation by activating subpopulations of stromal cells,
which then switch on the expression of chemokine genes.3 The
expression of CCR7 ligand attracts dendritic cells (DCs) and T
cells to form the T-cell area4; the expression of CXCR5 ligand
brings B cells together to form follicles.5 The genes for these
receptors, TNFR1 and LT␤R, are tightly linked on chromosome
12 in humans and chromosome 6 in mice, implying that they
arose by local gene duplication prior to speciation of human
and mouse.
The expression of the T-zone chemokines in lymph nodes is
normal in RAG⫺/⫺ mice, although the expression of the B-zone
chemokine, CXCL13, is reduced to approximately 20%, and
normal expression depends on B cells.6 Along with the LT␣⫺/⫺
lymphocyte transfer experiments, these data suggest that there is a
non-B non-T cell capable of inducing normal (T zone) and partial
(B zone) chemokine expression in stroma.
In this paper, we extend previous observations demonstrating a
role for a non-B non-T cell in B/T segregation,2 and identify
CD4⫹CD3⫺ cells that we have previously implicated in T-cell
memory for antibody responses in adult mice7,8 as playing a role in
the lymphoid stromal organization within secondary lymphoid
tissues. We report that adult CD4⫹CD3⫺ cells express high levels
of mRNA for LT␣, LT␤, tumor necrosis factor (TNF) ␣, and
LIGHT, which are the ligands for TNFR1 and the LT␤R. Levels of
expression are comparable with those expressed in embryonic and
neonatal CD4⫹CD3⫺ cells, and the expression of LT␤ is at least an
order of magnitude greater than in CD11c⫹ DCs or plasmacytoid
DCs (pDCs). Furthermore, using adoptive cell-transfer experiments, we demonstrate that the expression of these genes is
functional: fetal CD4⫹CD3⫺ cells derived from embryonic day (E)
15 spleen and adult CD4⫹CD3⫺ cells, but not lymphocytes, pDCs,
and DCs, are able to restore a significant degree of B/T segregation
in the spleens of LT␣⫺/⫺ mice, and up-regulate VCAM-1 and
CCL21 protein expression on the stroma.
Using confocal microscopy, we demonstrate that this
CD4⫹CD3⫺ cell associates closely with VCAM-1⫹ follicular
dendritic cells (FDCs) in B-cell areas as well as with the VCAM-1⫹
stromal population within the T zone.
Submitted April 24, 2006; accepted September 25, 2006. Prepublished
online as Blood First Edition Paper, October 3, 2006; DOI 10.1182/blood2006-04-018465.
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The publication costs of this article were defrayed in part by page charge
© 2007 by The American Society of Hematology
1602
Materials and methods
Mice
All experiments were performed in accordance with the United Kingdom
laws and with the approval of the University of Birmingham ethics
committee. Normal, RAG1⫺/⫺, LT␣⫺/⫺, and T-cell–deficient9 mice were
bred and maintained in our animal facility. Adult CD4⫹CD3⫺ cells were
isolated from RAG1⫺/⫺ mouse spleens from mice older than 6 weeks,
neonatal CD4⫹CD3⫺ cells were from 2-day-old normal C57Bl/6 or Balb/c
mouse spleens, and fetal (E15) CD4⫹CD3⫺ cells were taken from normal
C57Bl/6 or Balb/c embryo spleens from normal pregnant mice at gestation
day 15 and cultured with interleukin-7 (IL-7) for 5 to 7 days.
BLOOD, 15 FEBRUARY 2007 䡠 VOLUME 109, NUMBER 4
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BLOOD, 15 FEBRUARY 2007 䡠 VOLUME 109, NUMBER 4
Preparation of CD4ⴙCD3ⴚ cells, pDCs, DCs, and other cells
Cell suspensions for isolation of CD4⫹CD3⫺ cells, DCs, and pDCs were
made from the spleens of adult RAG⫺/⫺ mice as described previously.7,10
Briefly, CD11c⫹ cells were positively enriched by using CD11c-coated
magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany), and then
fluorescence-activated cell-sorter (FACS) sorted into CD8⫹ and CD8⫺
populations. CD4⫹ cells were enriched from CD11c⫹-depleted populations
using CD4-coated magnetic beads, and the resulting CD4⫹-enriched
populations sorted into CD4⫹CD3⫺B220⫺CD11c⫺ (CD4⫹CD3⫺) and
CD4⫹CD3⫺B220⫹CD11clow (pDC) populations. For Figure 6, CD4 enriched populations were prepared without CD11c⫹ cell depletion from
T-cell–deficient mice.
Adoptive cell transfer
Splenocytes (3 ⫻ 107) from either LT␣⫺/⫺ or normal mice were transferred
intraperitoneally into RAG⫺/⫺ hosts. Ten days after transfer the spleens of
the injected mice were taken and stained for confocal microscope analysis.
Adult CD4⫹CD3⫺ cells (1.6 ⫻ 105) or 1.7 ⫻ 105 E15 CD4⫹CD3⫺ cells
or 1 ⫻ 106 CD11c⫹ DCs and pDCs from RAG⫺/⫺ mice or 4 ⫻ 106
splenocytes from normal mice were transferred intravenously into LT␣⫺/⫺
recipient mice. Each cell population was transferred into 2 mice per
experiment, and at least 2 experiments were performed. Ten days after
transfer the spleens of the injected mice were taken and stained for confocal
microscope analysis.
TaqMan low-density array analysis
TaqMan primer sets (Applied Biosystems, Warrington, United Kingdom) are
designed to work with an efficiency approaching 100%, enabling the quantitative
comparison of mRNA expression for different genes. Housekeeping genes
(␤-actin in these experiments) were used to correct for total mRNA (the
level to which the ␤-actin signal was corrected in all mRNA samples). For
Figure 3, TNF␣, LT␤, LIGHT, and ␤-actin from the sets were analyzed.
FACS staining
CD4-enriched cells were stained for anti-CD11c FITC, anti-CD4 PE, and
anti-B220 allophycocyanin monoclonal antibodies (mAbs) with biotinylated mAbs against OX40L and CD30L (BD Biosciences, Palo Alto, CA) in
conjunction with streptavidin cychrome (BD Biosciences) as the secondstep staining reagent. To stain with LT␤R–immunoglobulin (Ig) or
control-Ig fusion protein, CD4⫹CD3⫺ cells cultured in the presence of
100 ng/mL IL-7 for 7 days prior to staining with LT␤R-Ig (a kind gift of
Dr Jeff Browning, Biogen, Cambridge, MA). Second-step reagent was
goat anti–human IgG FITC (Jackson Immunoresearch Laboratories,
West Grove, PA).
Immunohistology for confocal microscope
Confocal images were acquired using a Zeiss Axiovert 100M microscope
(Zeiss, Welwyn Garden City, United Kingdom) equipped with a capochromat 10⫻/0.45 numerical aperture (NA) or a c-apochromat 63⫻/1.2
NA water objective. Comparison of lymphocyte architecture in spleens
from wild-type and variously treated or gene-modified mice was performed
using Zeiss LSM510 (laser scanning microscopy) software on confocal
micrographs taken from sections stained for IgM and CD3 as described
previously,7 in conjunction with staining with fluoresceinated anti–
VCAM-1 mAb (BD Biosciences) at a previously optimized dilution. The
intense staining of VCAM-1⫹ cells in splenic red pulp contrasted with less
intense and patchier appearance of VCAM-1 staining in white pulp areas.
We therefore used the limits of the VCAM-1hi staining as a lymphocyteindependent indicator of the extent of the white pulp areas. Intuitively, if B
cells are stained with 1 fluorochrome and T cells with another, when there is
increased B/T segregation, then there will be less overlapping of colors.
This is what the following objective algorithm was designed to test.
Regions of white pulp delineated by red pulp VCAM-1 expression were
extracted and the area (square micrometers) and total pixels determined by
Zeiss confocal software were recorded. Within the extracted region we
CD4⫹CD3⫺ CELLS ASSOCIATE WITH B- AND T-ZONE STROMA
1603
enumerated the pixels registering intensity for IgM (B cells) and CD3
(T cells). Where B cells and T cells are within 0.25 ␮m (the pixel dimension
using the ⫻ 25 objective) of one another, the pixels are recorded as double
positive. We quantified double positivity by multiplying the IgM and CD3
matrices of the micrographs together to produce a separate array
(IgM⫹CD3⫹) that we used as a measure of the degree of contact between
B and T cells. Numbers of singly positive pixels (IgM or CD3) were
determined by subtracting the numbers of IgM⫹CD3⫹ pixels from those in
the respective original arrays. Pixel number (for each magnification on the
confocal microscope there is a fixed relationship between pixel number and
area in square micrometers) was then used to provide an estimate of the
areas within each white pulp region taken up by B-cell or T-cell membrane,
or both (IgM⫹, CD3⫹, and IgM⫹CD3⫹, respectively).
To quantify the VCAM-1 expression in T zones (shown in Figure 4B),
T-cell areas were delineated and the number of VCAM-1–positive pixels
were enumerated, both using Zeiss confocal software.
Spleen sections were examined systematically for all identifiable areas
of white pulp; routinely, 10 different areas were photographed per spleen.
Following statistical evaluation, median values with ranges for each
treatment were selected for display purposes.
For detection of chemokine expression, sections were first blocked with
10% horse serum for 10 minutes, then stained with polyclonal IgG goat sera
directed against mouse CCL19, mouse CCL21, or mouse CXCL13 (R&D
Systems, Minneapolis, MN) for 50 minutes at room temperature, at
concentrations determined by titration. The Abs were detected using
donkey anti–goat IgG Cy2 (Jackson Immunoresearch Laboratories) preabsorbed with 10% horse serum.
Results
Evidence that TNFR and LT␤R signals from a non-B non-T cell
play a critical role in B/T segregation
Many groups have demonstrated that continuous LT␤R signals are
required to maintain B/T segregation and B follicles.3,11 We
confirmed these findings by acutely blocking LT␤R signals (data
not shown). Injection of LT␤R-Ig was associated with rapid loss of
discrete B follicles and VCAM-1⫹ FDCs, as reported by others.12
LT␣⫺/⫺ mice, which also have low levels of TNF␣13 and are
therefore deficient in both TNFR1 and LT␤R signals, show an even
greater degree of disorganization of lymphocytes (Figure 1),
confirming that these 2 signaling pathways have independent
effects on lymphoid organization.14,15
Although lymphocytes, particularly B cells, express both
TNFR1 and LT␤R ligands (Figure 2),1 injection of normal
splenocytes into LT␣⫺/⫺ mice failed to restore a B/T-segregated
architecture (Figure 1Aiii) as reported previously.2 RAG⫺/⫺
mice lack B and T cells, but other cell types are present. To test
whether the environment within RAG⫺/⫺ mice was capable of
segregating B and T cells, splenocytes from LT␣⫺/⫺ mice were
transferred into RAG⫺/⫺ mice. Ten days after cell transfer, the
reconstituted spleens were examined for lymphocyte architecture. Whether reconstituted with LT␣⫺/⫺ (Figure 1Aiv) or
normal (data not shown) lymphocytes, RAG⫺/⫺ spleens demonstrated clear B/T segregation by comparison with spleens from
LT␣⫺/⫺ mice (Figure 1Aiv).
Adult CD4ⴙCD3ⴚ and embryonic/neonatal CD4ⴙCD3ⴚ cells, but
not pDCs or DCs, express high levels of LT␤ and TNF␣
To investigate which non-B non-T cells in adult tissue expressed LT␣, LT␤, and TNF␣, we used TaqMan polymerase
chain reaction (PCR) analysis on purified subpopulations:
CD8⫹CD11c⫹B220⫺ and CD8⫺CD11c⫹B220⫺ DCs, pDCs
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1604
KIM et al
BLOOD, 15 FEBRUARY 2007 䡠 VOLUME 109, NUMBER 4
Figure 1. Regulation of B/T segregation. (A) Low-magnification confocal images of spleen sections. CD3 is shown in red and IgM in green. Yellow shows colocalization of B
and T cells. (i) Wild-type (WT) mice. (ii) LT␣⫺/⫺ mice. (iii) Organization of the spleens of LT␣⫺/⫺ mice 10 days after transfer of normal splenocytes. (iv) Organization of the
spleens of RAG⫺/⫺ mice 10 days after transfer of LT␣⫺/⫺ splenocytes. (v) Organization of the spleens of LT␣⫺/⫺ mice 10 days after transfer of CD11c⫹-enriched population (DCs
and pDCs). (vi) Organization of the spleens of LT␣⫺/⫺ mice 10 days after transfer of CD4⫹CD3⫺ cells. (B) CCL21 expression. CD3 is shown in red, IgM in green, and CCL21 in
white. Single staining for CCL21 is shown in the adjacent box. Scale bars represent 100 ␮m. Results representative of at least 2 separate experiments.
(CD4⫹CD11clowB220⫹) and the CD4⫹CD3⫺ accessory cells that
we have recently characterized (CD4⫹CD3⫺CD11c⫺B220⫺)
from adult RAG⫺/⫺ mice,7,8 as well as CD4⫹CD3⫺CD11c⫺
B220⫺cells from embryonic (E15) and neonatal (day [D] 2)
spleens. Results are shown in Figure 2A: levels of TNF␣, LT␤,
and LIGHT (also a LT␤R ligand) were comparable in all 3
populations of CD4⫹CD3⫺CD11c⫺B220⫺ cells, as was the
expression of LT␣ (determined by conventional semiquantitative PCR; data not shown). In contrast, the expression of TNF␣,
LT␤, and LIGHT on pDC, natural killer (NK) cell, or DC
subpopulations was at least an order of magnitude less. T and B
cells expressed high levels of LT␤R ligands, but much lower
levels of TNF␣ than either embryonic/neonatal or adult
CD4⫹CD3⫺ cell populations.
To confirm that CD4⫹CD3⫺ cells expressed LT␤R ligands at
the protein level, we stained CD4⫹CD3⫺ cells with a LT␤R-Ig
fusion protein. Although this reagent stains freshly isolated
CD4⫹CD3⫺ cells, following in vitro culture in the presence of IL-7,
LT␤R ligands were clearly up-regulated on CD4⫹CD3⫺ cells
(Figure 2B).
CD4ⴙCD3ⴚ cells but not splenocytes are capable of organizing
lymphocytes in LT␣ⴚ/ⴚ mice
The strong expression of both TNFR1 and LT␤R ligands on both
embryonic/neonatal and adult CD4⫹CD3⫺ cells suggested that they
might be capable of organizing LT␣⫺/⫺ lymphocytes. To test this
directly, CD4⫹CD3⫺ cells from RAG⫺/⫺ mice were transferred intravenously (or in some experiments intraperitoneally) into LT␣⫺/⫺ recipient
mice. Ten days after transfer there was evidence of B/T segregation in
LT␣⫺/⫺ mice (Figure 1Avi). A montage of 6 confocal images taken of
LT␣⫺/⫺ spleen sections with a ⫻ 10 objective is shown without (Figure
3A) and following (Figure 3B) reconstitution with CD4⫹CD3⫺ cells. In
contrast, mice that received CD11c-enriched fractions (CD11c⫹ DCs
and CD11clow pDCs; Figure 1Av) or splenocytes (Figure 1Aiii) showed
little evidence of B/T segregation.
Figure 2. mRNA expression of TNFR1 and
LT␤R ligands on CD4ⴙCD3ⴚ and other
cell populations. (A) mRNA for the genes
indicated were assayed by quantitative PCR
and corrected for ␤-actin expression as described in “Materials and methods.” Bars
show standard deviations for 4 separate
experiments. (B) Staining of CD4⫹CD3⫺ cells
with LT␤R-Ig. Isolated CD4⫹CD3⫺ cells were
cultured in the presence of 100 ng/mL IL-7
prior to staining with LT␤R-Ig (䡺) or control-Ig fusion protein (u). Of live gated
CD4⫹CD3⫺ cells, 55.2% expressed LT␤R
ligands.
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BLOOD, 15 FEBRUARY 2007 䡠 VOLUME 109, NUMBER 4
CD4⫹CD3⫺ CELLS ASSOCIATE WITH B- AND T-ZONE STROMA
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Figure 3. Montage of low magnification confocal images of
spleen sections of LT␣ⴚ/ⴚ mice. (A) LT␣⫺/⫺ mice. (B) Organization of the spleens of LT␣⫺/⫺ mice 10 days after transfer of
CD4⫹CD3⫺ cells. CD3 is shown in red and IgM in green. Yellow
shows colocalization of B and T cells. Inset boxes show single
staining for VCAM-1 (in white) in the T zones as outlined by white
figures in main pictures. Scale bar represents 100 ␮m. Results
representative of at least 2 separate experiments.
To detect whether chemokine expression was up-regulated in
LT␣⫺/⫺ spleens after cell transfer, CCL19 and CCL21 (T-zone
chemokines) and CXCL13 (B-zone chemokine) were stained as
described.16 Wild-type spleens showed strong CCL21 expression in
T zones (Figure 1Bi) compared with LT␣⫺/⫺ spleens (Figure 1Bii).
Following reconstitution with adult CD4⫹CD3⫺ cells or E15
CD4⫹CD3⫺ cells (Figure 1Bv-Bvi), CCL21 expression in LT␣⫺/⫺
spleens was clearly up-regulated in areas where T cells are
segregated from B cells. In contrast, LT␣⫺/⫺ spleens that received
either splenocytes (Figure 1Biii) or CD11c-enriched cells (Figure
1Biv) did not show increased CCL21 expression. CCL19 expression was much weaker than CCL21 expression in spleens from
normal mice, and was not detected in LT␣⫺/⫺ spleens before or
after cell transfer (data not shown). Although CXCL13 was
strongly expressed in normal B follicles, its expression was not
detected in LT␣⫺/⫺ spleens before or after cell transfer (data not
shown). Furthermore, FDC markers were not induced (data not
shown), consistent with previous reports that B-cell expression of
LT␤R ligands is critical for CXCL13 expression.3
As described in “Materials and methods” for each experiment,
areas of splenic white pulp from reconstituted LT␣⫺/⫺ mice were
analyzed to identify the degree of B/T segregation: discrete red (T
cell) and green (B cell) areas versus yellow areas (mixed).
Compared with control LT␣⫺/⫺ spleen sections, white pulp areas
containing B and T lymphocytes were significantly larger in mice
reconstituted with adult CD4⫹CD3⫺ cells (P ⫽ .005) and E15
CD4⫹CD3⫺ cells (P ⫽ .001), but not CD11c⫹ populations (P ⫽ .07)
or splenocytes (P ⫽ .28) (Figure 4Ai). This was due to significantly
increased T-cell–free B-cell areas for adult CD4⫹CD3⫺ cells
(green; P ⫽ .005) and E15 CD4⫹CD3⫺ cells (P ⫽ .002), but not
CD11c⫹ cells (P ⫽ .6) or splenocytes (P ⫽ .28) (Figure 4Aii). The
B-cell–free T-cell areas (red) were also significantly bigger (almost
twice as large) in the spleens transferred with adult CD4⫹CD3⫺
cells (P ⫽ .008) and E15 CD4⫹CD3⫺ cells (P ⫽ .004) and CD11c⫹
cells (P ⫽ .04), but not splenocytes (P ⫽ .39) (Figure 4Aiii).
The hypothesis that CD4⫹CD3⫺ cells caused B/T segregation
was supported by evidence demonstrating significant up-regulation
of VCAM-1 in T-cell stroma in LT␣⫺/⫺ mice injected with both
embryonic and adult CD4⫹CD3⫺ cells (Figures 3, 4B). Analysis
showed adult CD4⫹CD3⫺ cells (P ⫽ .001) and E15 CD4⫹CD3⫺
cells (P ⫽ .004) but not CD11c⫹ cells (P ⫽ .25) or splenocytes
(P ⫽ .68) (Figure 4B) induced significantly more VCAM-1 expression in T-cell areas.
Although by confocal analysis we detected increased B/T
segregation in CD4⫹CD3⫺-cell–injected mice, we did not detect
up-regulation of mRNA for the homeostatic chemokines CCL19,
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KIM et al
BLOOD, 15 FEBRUARY 2007 䡠 VOLUME 109, NUMBER 4
Figure 4. Splenic areas and VCAM-1 expression after adoptive transfer. (A) Areas of splenic white pulps, B cells, and T cells. White pulp area (i), B-cell area (ii), and T-cell
area (iii) in LT␣⫺/⫺ mice that were adoptively transferred with normal splenocytes, CD11c⫹ DCs and pDCs, adult CD4⫹CD3⫺ cells, or E15 CD4⫹CD3⫺ cells compared with
normal white pulp areas. Bar shows mean values with high and low values calculated from 10 different white pulp areas. (B) VCAM-1 expression after adoptive transfer.
Statistical differences were calculated using a nonparametric Mann-Whitney test. *Statistical significance by Mann-Whitney P ⱕ .05; **P ⱕ .005. Results representative of at
least 2 experiments.
CCL21, and CXCL13 (data not shown). We propose this is because
the small numbers of donor CD4⫹CD3⫺ cells that reach the
recipient spleen are unable to raise the total homeostatic chemokines levels above background, but that focal increases in chemokine expression account for the observed increased degree of B/T
segregation observed.
Although VCAM-1 expression was up-regulated by CD4⫹CD3⫺
cells, we did not observe up-regulation of the T-zone stromal
marker, gp38,17 or expression of MadCAM-1,18 which stains the
marginal sinus of normal mice but not LT␣⫺/⫺ splenic tissue. We
also stained for CR1 (CD35) and the FDC markers FDC-M1 and
FDC-M2. Although there was up-regulation of CD35 by all
populations transferred into LT␣⫺/⫺ mice, no population (including
B and T splenocytes or CD4⫹CD3⫺ cells) up-regulated either
FDC-M1 or FDC-M2 (data not shown).
tent with rapid loss of VCAM-1⫹–expressing FDCs (data
not shown).12
To examine the relationship between CD4⫹CD3⫺ cells and
the VCAM-1⫹ stromal cells, we examined sections of splenic
tissue from T- and NK-cell–deficient mice,9 where the absence
of CD4⫹CD3⫹ T cells in the DC-rich areas makes it straightforward to identify other CD4⫹ cells (Figure 5C). As reported
previously,7 we found CD4⫹CD3⫺ cells in B follicles, and these
were closely associated with VCAM-1⫹ cells (Figure 5C [low
magnification]–D [high magnification]). However, in the T- and
NK-cell–deficient mice we were also able to identify a similar
association between a CD4⫹CD3⫺ population and the local
VCAM-1⫹ population among the CD11c⫹ DCs in the area
populated by T cells in normal mice (Figure 5C [low magnification], E [high magnification]).
CD4ⴙCD3ⴚ cells are closely associated with VCAM-1ⴙ stromal
cells in B- and T-cell areas
CD4ⴙCD3ⴚ cells interact with CD11cⴙ DCs
In the developing embryo, lymphoid tissue inducer (LTi) cells
interact with stromal cells in lymph node anlagen to up-regulate
the expression of the chemokines that recruit lymphocytes to
form lymph nodes,19 and there is evidence that LT␣ and LT␤
expressing LTi cells are responsible for B/T segregation in the
neonatal lymph node.20 Effective delivery of LT␤R signals to
the stromal cells involves interactions between the LTi-cell
integrin, ␣4␤1, and its ligand, VCAM-1, which is expressed on
stroma.21 We reasoned that adult CD4⫹CD3⫺ cells would
function in a similar way. To examine their relationship with
VCAM-1⫹ stromal cells we first identified VCAM-1⫹ populations in normal adult mice. The red pulp of the spleen exhibits
strong staining for VCAM-1⫹ cells, but there is also generally
less intense staining in white pulp areas, with discrete staining in
both B follicles (including FDC populations) and T-cell areas
(Figure 5A). Comparable with normal mice, LT␣⫺/⫺ mice show
strong staining for VCAM-1 in the red pulp of the spleen, but
VCAM-1 staining is largely missing from white pulp areas,
which, although lymphocyte rich, show no segregation of B and
T cells (Figure 5B). In mice injected with LT␤R-Ig, where LT␤R
but not TNFR1 signals are blocked, VCAM-1 expression is
maintained in both T zone and red pulp areas, but there is
selective loss of VCAM-1 expression within B follicles consis-
To ensure that the CD4⫹CD3⫺ cell population that we identified in
the T zone was not CD4⫹ DCs or pDCs, we carefully characterized
all the CD4⫹ populations from T-cell– and NK-cell–deficient
mice.9 Four CD4⫹ populations could be identified from these mice.
The first is the CD4⫹CD3⫺ cell population which we have
characterized previously,7 which lacks expression of B220 and
CD11c but which expresses high levels of OX40L and CD30L
(Figure 6A). The second is the pDC population, which expresses
B220 and low levels of CD11c but lacks expression of OX40L and
CD30L (Figure 6B), and the third is the CD4lowCD11c⫹ myeloid
DC population, which expresses low levels of OX40L but not
CD30L (Figure 6D). Whereas the first 3 populations are homogeneous with respect to size, the fourth population (CD4⫹CD11c⫹) is
heterogeneous, being divisible by differential forward scatter into 2
subpopulations (Figure 6C). The population of smaller cells closely
resembles classic myeloid DC phenotype cells, whereas the
population of larger cells has the appearance of clusters of
CD4⫹CD3⫺ cells and myeloid DCs: its phenotype is mixture of the
phenotypes of these 2 cell types. Although the clusters had
apparently slightly higher levels of B220, we think this is a result of
the increased fluorescence of larger objects. These data support the
idea that there is some association between CD4⫹CD3⫺ populations and DCs, confirming the impressions formed from our
confocal microscopy.
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BLOOD, 15 FEBRUARY 2007 䡠 VOLUME 109, NUMBER 4
CD4⫹CD3⫺ CELLS ASSOCIATE WITH B- AND T-ZONE STROMA
1607
Figure 5. Evidence that CD4ⴙCD3ⴚ cells are associated with VCAM-1ⴙ stromal cells. Confocal images of spleen sections showing (A) wild-type (WT) and (B) LT␣⫺/⫺ mice.
Dotted yellow area of panel A identifies B follicles in WT mice. To show association of VCAM-1⫹ cells with CD4⫹CD3⫺ cells, confocal images from T-cell–deficient mice were
analyzed at low magnification (10⫻) (C), high magnification (63⫻) of B-cell area (D), and high magnification of T-cell area (63⫻) from panel C (E). Scale bar represents 50 ␮m
for low magnification, 20 ␮m for high magnification.
Many CD4ⴙCD3ⴚ cells identified in T-cell areas are not pDCs or
DCs, and are also found adjacent to central arterioles in spleen
and PNAdⴙ HEVs in the lymph nodes
To exclude the possibility that CD4⫹CD3⫺ cells identified in T-cell
areas were pDCs or DCs, we stained sections from mice deficient in
T and NK cells,9,22 excluding CD3, CD11c, and B220, using
FITC-conjugated antibodies (green) and counterstaining with CD4
(red) (Figure 7A). Although some CD4⫹ cells expressed either
CD11c or B220 (yellow), there were also many red-only cells,
which therefore express neither B220 nor CD11c, providing direct
confirmation that CD4⫹CD3⫺ cells are located in the T zone.
Figure 6. CD4ⴙ populations in the spleen.
CD4⫹ populations were isolated from T- and
NK-cell–deficient mice and characterized for
expression of CD11c, B220, and OX40L and
CD30L. Shaded histograms show control
staining. Results representative of 4 experiments.
Furthermore, the red-only cells are often found adjacent to yellow
cells, consistent with the possibility that CD4⫹CD11c⫹ DCs and
CD4⫹CD3⫺ cells associate. The location of CD4⫹CD3⫺ cells in
the T-cell areas was not an artifact of T-cell–deficient mice, as
careful analysis of normal mouse spleens revealed CD4⫹CD3⫺
cells within the T-cell areas (Figure 7B). These data indicate that
CD4⫹CD3⫺ cells associate with stroma in both B- and T-cell areas
and are therefore well positioned to provide the TNFR1 and LT␤R
signals that induce chemokine expression. Furthermore, like LTi
cells, which are found clustered around blood vessels in neonatal
spleens,23 CD4⫹CD3⫺ cells are also found around central arterioles
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1608
KIM et al
BLOOD, 15 FEBRUARY 2007 䡠 VOLUME 109, NUMBER 4
Figure 7. CD4ⴙCD3ⴚ cells found around
the central arteriole in the spleen and
associated with PNAdⴙ HEVs in the lymph
node. Confocal images of T-cell–deficient
mouse spleens stained with B220 and CD11c
in green, and CD4 in red (A), and normal
mouse spleens stained with CD3, B220, and
CD11c in green and CD4 in red (B). Area
indicated by closed yellow line identifies
central arteriole in high magnification images
of panels A and B. (C) Confocal images of
lymph node from a normal mouse stained
with CD3, B220, and CD11c in green, CD4 in
red, IgM in gray, and PNAd in blue. Scale bar
represents 100 ␮m for low magnification
(10⫻), and 20 ␮m for high magnification
(63⫻).
in the spleen (Figures 7A-B) and in the lymph nodes are found
associated with peripheral lymph node addressin–positive (PNAd⫹)
high endothelial venules (HEVs; Figure 7C).
Discussion
The adult CD4⫹CD3⫺ cell in secondary lymphoid tissues constitutively expresses the TNF family members OX40L and CD30L.7,8,24
It provides the signals through OX40 and CD30 on activated CD4
T cells that (1) maintain them as follicular T cells that select B cells
in affinity maturation within germinal centers; and (2) support them
as the memory T cells that help secondary B-cell responses. Here
we demonstrate that adult CD4⫹CD3⫺ cells, like the embryonic
LTi cells with which they share a common phenotype,7,24 express
high levels of a second set of TNF family genes, LT␣, LT␤, and
TNF␣, which are linked with the organized B/T segregation
observed in lymphoid tissues.1 Several pieces of evidence support
the role of these cells in B/T segregation. First, we show that the
LT␣⫺/⫺ lymphocytes that are unable to segregate in their parental
environment segregate normally in RAG-deficient hosts, which
lack mature B and T cells. When we subfractionated RAG-deficient
accessory cells and transferred them into LT␣⫺/⫺ hosts, we
identified CD4⫹CD3⫺ cells as cells capable of the segregation of
lymphocytes into B- and T-cell areas, whereas splenocytes (including B and T cells) or CD11c⫹ DC and pDC populations failed to do
so to a significant extent.
Evidence that CD4⫹CD3⫺ cells were in a position to elicit the
secretion of homeostatic chemokines from stromal populations was
provided by the demonstration of their tight association with
VCAM-1⫹ stromal cells in both T- and B-cell areas. Furthermore,
transfer of CD4⫹CD3⫺ cells into LT␣⫺/⫺ recipients up-regulated
VCAM-1 and CCL21 expression on host T-zone stromal cells in
areas where T cells were segregated from B cells, a situation similar
to that reported for the LTi-cell–induced expression of VCAM-1 in
lymph node anlage.19 The conclusion that CD4⫹CD3⫺ cells are
important for organizing lymphoid tissues is further supported by
observations that lymph node expression of CCL19 and CCL21 is
not dependent on B or T cells.6 Although expression of CXCL13
does appear to substantially depend on B cells,6 we found that
normal splenocytes alone were not sufficient to induce either FDC
markers or CXCL13 expression in LT␣⫺/⫺ mice. We propose that
CD4⫹CD3⫺ cells might be essential for B follicle formation
because they express a critical signal not expressed by B cells, and
that the 2 cell types might act synergistically together in B follicle
formation. A possible candidate for such a signal is TNF␣, which is
expressed at much higher levels on CD4⫹CD3⫺ cells than on B
cells, and which is essential for the formation of primary B follicles
and FDC networks.25
Therefore, the data reported previously and here link CD4⫹CD3⫺
cells with 2 functions: T-cell survival via OX40 and CD30
signals,7,8,24 and with the activation of stromal cells that produce
homeostatic chemokines. We propose that CD4⫹CD3⫺ cells help to
establish the chemokine gradients that guide the migration of B and
T cells to their respective locations by activating stromal cells to
up-regulate CCR7 and CXCR5 ligands. In addition, we propose
that CD4⫹CD3⫺ cells enable germinal center (GC) T-cell selection
of B cells by excluding CCR7⫹ T cells from B follicles, while
allowing locally primed CXCR5⫹ T cells into B follicles. Within
B-cell GCs, the CD4⫹CD3⫺ cells are attached both to FDCs and to
the CXCR5⫹ T cells that select GC B cells.7,8 We suggest that they
act as a tether between the selecting T cells and the FDCs, thus
forming the microenvironment for B-cell selection that is essential
for affinity maturation.
In the outer T zone, CD4⫹CD3⫺ cells interact with both T-zone
stroma and primed CD4 T cells. Memory B and T cells depend on
the cues established by homeostatic chemokines expressed in Band T-zone stroma to guide their interactions to the B/T interface.
Although T-cell priming does not depend on LT␣-dependent B/T
segregation, B/T collaboration for memory antibody responses
does.26 The observation that “balanced” expression of CXCR5 and
CCR7 on antigen-activated B cells27 and CD4 T cells28 locates
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BLOOD, 15 FEBRUARY 2007 䡠 VOLUME 109, NUMBER 4
CD4⫹CD3⫺ CELLS ASSOCIATE WITH B- AND T-ZONE STROMA
them at the B/T interface suggests a mechanism whereby B- and
T-zone chemokine signals regulate B/T interactions during secondary antibody responses. We propose that the outer T zone therefore
forms the microenvironment for memory responses: OX40- and
CD30-deficient mice have grossly impaired memory antibody
responses because of failure of T cells to survive in this location,7,8
which is where memory T and B cells interact during secondary
responses.29
We also found evidence for an association between DCs and
CD4⫹CD3⫺ cells. CD4⫹CD3⫺ cells express LT␤R ligands implicated in homeostatic signaling to the CD4⫹ myeloid DCs30
associated with the development of T helper 2 (Th2) antibody
responses.31 In addition, CD4⫹CD3⫺ cells constitutively express
TRANCE (TNFSF11),7 which can signal survival through its
ligand RANK (TNFRSF11A), expressed on DCs.32 TRAF6, a key
target for RANK signaling, is essential for CD4⫹ DCs.33 Our data
are therefore consistent with the view that CD4⫹CD3⫺ cells attach
to stroma at the B/T interface and activate their expression of the
chemokines that recruit both myeloid DCs and primed T cells.
Once recruited, DCs and T cells would then be maintained by
combinations of TNF survival signals and homeostatic chemokines, provided by the CD4⫹CD3⫺/stromal association.
Our hypothesis that CD4⫹CD3⫺ cells play a role in segregating
B and T cells in normal lymphoid tissue is at odds with the
observation that mice deficient in the splice variant of the retinoic
acid orphan receptor (ROR␥t) lack LTi cells. While these mice fail
to develop lymph nodes and gut-associated lymphoid tissue, their
spleens are normally segregated into B- and T-cell areas.34,35
However, we have examined spleens from ROR␥t⫺/⫺ mice and
found, at least by confocal microscopy, that CD4⫹CD3⫺ cells are
present, indicating that splenic CD4⫹CD3⫺ cell populations might
not depend on ROR␥t expression (F.M.M. and P.J.L.L., unpublished observations, December 2004). In support of this proposition, ROR␥t⫺/⫺ mice also have nasal-associated lymphoid tissue
(NALT),36 and there is evidence that CD4⫹CD3⫺ cells are required
for NALT formation.37 Expression of ROR␥t has been primarily
linked with survival signals to CD4⫹CD3⫺ cells,34,38 so it might
only be required for their survival in lymph node anlage but not in
the spleen and NALT.
The function of CD4⫹CD3⫺ accessory cells in forming microenvironments for adaptive T-cell responses is possibly relevant to
the pathogenesis of AIDS in HIV infection. The expression by
1609
CD4⫹CD3⫺ cells of CXCR4,39 which is associated with the
development of normal GCs,40 may be the link between the
emergence of CXCR4-tropic variants of HIV and progressive
disease.41-43 HIV localizes on FDCs,44 with which CD4⫹CD3⫺
cells are directly associated; therefore, they are potentially susceptible to infection. Destruction of CD4⫹CD3⫺ cells, either by
CXCR4-tropic HIV variants or, more likely, by the host CD8
immune response that invades B follicles,45 predicts the signature
of progressive disease: loss of the discrete B follicle structure
(follicular fragmentation and lysis), disruption of the FDC network,46 and loss of the functional capacity to make de novo
high-affinity antibody responses.47 The impairment of the capacity
to mount neutralizing antibodies is associated with rising viral
titers and loss of T cells from the T zone. We propose that the
decline in T-cell numbers in humans might be due at least in part to
loss of T-zone CD4⫹CD3⫺ cells, with consequent loss of CCL19
and CCL21, a view supported by the loss of these chemokines in a
simian model of AIDS.48 Finally, our hypothesis provides an
explanation for the loss of HEVs in terminal AIDS.49
Acknowledgments
We would like to thank Jeff Browning (Biogen) for providing
LT␤R-Ig.
Supported by a Wellcome Programme Grant to P.J.L.L.
Authorship
Author contributions: M.-Y.K., F.M.M., G.A., and P.J.L.L. designed and performed the research, collected and analyzed the data,
and wrote the paper; F.M.M., F.M.C.G., A.W., S.H.G., and V.B.
performed the research and confocal picture analysis; and L.S.K.W.,
J.C., and E.J. contributed to performing the research.
Conflict-of-interest statement: The authors declare no competing financial interests.
Correspondence: P. J. L. Lane, MRC Centre for Immune
Regulation, Institute for Biomedical Research, Birmingham Medical School, Birmingham B15 2TT, United Kingdom; e-mail:
[email protected].
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2007 109: 1602-1610
doi:10.1182/blood-2006-04-018465 originally published
online October 3, 2006
Function of CD4+CD3− cells in relation to B- and T-zone stroma in
spleen
Mi-Yeon Kim, Fiona M. McConnell, Fabrina M. C. Gaspal, Andrea White, Stephanie H. Glanville,
Vasilios Bekiaris, Lucy S. K. Walker, Jorge Caamano, Eric Jenkinson, Graham Anderson and Peter J.
L. Lane
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