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*
Centre Hospitalie Université de Montreal Research Centre, University of Montreal, Montreal, Quebec, Canada;
Fujisaki Institute, Hayashibara Biochemical Laboratories, Okayama, Japan; Department of Bioregulatory Function, University of Tokyo, School of Medicine, Tokyo, Japan; and
Department of Obstetrics and Gynecology, University of Montreal, Montreal, Quebec, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(LT3), as
determined by ELISA, RNase protection assay, and intracytoplasmic
staining. Besides LT3 and IL-2, these cells also produce high levels
of TNF- together with significant amounts of IFN-
and IL-13.
Naive cells also express LTß mRNA and the membrane form of LT
(LTß). On average, naive CD4+ T cells secrete four
times more LT3 than Th1-like cells, twice more than naive
CD8+ T cells, and ten times more than B cells. Thus, naive
T cells express a large spectrum of cytokines, mainly of the Th1 type,
and the very high levels of LT3/TNF- that they release may play
an hitherto unsuspected role in the early stage of T cell-dependent
immune responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA
and trigger IgG2a switching in the T cell zone of lymphoid organs (9).
In addition to IL-4, naive human CD4+ T cells were reported
to release low levels of IFN- and IL-13 during primary activation in
vitro (7, 10). In agreement with the notion that the activation of
naive T cells is more dependent upon costimulatory signals than that of
effector/memory cells, the production of cytokines other than IL-2 by
naive T cells was found to require optimal CD28-mediated costimulation
(3, 6, 11). Here, we report the unexpected finding that naive human
CD4+ T cells are capable of producing very high levels of
soluble lymphotoxin (LT)5 (LT3), a
typical Th1 cytokine that is also known to play an essential role in
the organogenesis of secondary lymphoid organs (12). Besides IL-2 and
LT3, anti-CD3/B7-activated naive CD4+ lymphocytes
also release high levels of TNF- together with moderate amounts of
IFN- and IL-13, indicating that their cytokine repertoire is broader
than previously thought. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti-CD3 mAb (UCHT-1) was kindly provided by Dr. P. Beverley
(University College Middlesex School of Medicine, London, U.K.).
Neutralizing mouse anti-human IL-4 mAb (clone 8F12), recombinant
human (h) granulocyte-macrophage CSF, rhIL-2, and
rhIL-12 were kindly provided by Drs. C. Heusser (Novartis, Basel,
Switzerland), D. Bron (Bordet Institute, Brussels, Belgium), F. K.
Kahn, and M. Gately (Hoffmann-La Roche, Nutley, NJ), respectively.
Soluble CD40L trimer and rhIL-4 were received from Immunex (Seattle,
WA). rhTNF- was purchased from Genzyme (Cambridge, MA). The CD32 and
B7.1 double-transfected mouse L fibroblasts have been previously
described (6).
Lymphocyte preparation and culture conditions
Highly purified CD4+ and CD8+ T cells
were isolated from umbilical cord blood of healthy neonates, exactly as
described (7). The resulting populations were >98% viable, >98%
CD3+, CD4+/CD8-, or
CD4-/CD8+ and CD45RA+. They
contained no detectable CD45ROhigh, CD25+,
CD19+, and CD16+ cells. B cells were isolated
from cord blood mononuclear cells by depletion of E-rosette-forming
cells, followed by treatment with L-Leucine methyl ester
and lympho-kwik B (One Lambda, Canoga Park, CA). Adult naive
CD4+ T cells, defined as
CD45RO-CD31+, were isolated by cell sorting
(FACSort; Becton Dickinson, Mountain View, CA) after staining the
CD4+ T cells with FITC-conjugated anti-CD31 and
phycoerythrin-conjugated anti-CD45RO mAbs. T cells (1 x
106/ml) were cultured in 48-well culture plates in 0.5 ml
of RPMI 1640 medium containing 10% FCS, 5 mM L-glutamine,
50 IU penicillin G, and 50 µg streptomycin and were stimulated with
anti-CD3 mAb (200 ng/ml) immobilized on mitomycin-C-treated
CD32/B7.1 L cells (0.25 x 106/ml). For the induction
of Th2 and Th1 polarized cells, T cells were cultured in the presence
of rIL-4 (10 ng/ml) or a combination of rIL-12 (50 pM) and
anti-IL-4 mAb (10 µg/ml), respectively. After 3 days,
cells were washed and cultured at 0.25 x 106/ml in
culture medium supplemented with 50 U/ml rhIL-2 in 24-well plates.
After 4 days of IL-2 expansion, cells were washed and restimulated with
anti-CD3 mAb and CD32/B7.1 L cells. B cells (1 x
106/ml) were stimulated as indicated for 2 days. In some
experiments, CD4+ T cells were stimulated with irradiated
allogeneic dendritic cells for 5 days and expanded in IL-2 for an
additional 5 days. Dendritic cells were derived from adult
blood monocytes cultured for 7 days with IL-4, granulocyte-macrophage
CSF, and TNF-, exactly as described (7, 8).
Cytokine measurement
IL-2, LT3, TNF-, and IFN- were measured by sandwich
ELISA and radioimmunoassay, as previously described (7, 13).
IL-13 was measured with the Quantikine ELISA kit (R&D Systems,
Minneapolis, MN).
RNase protection assay
After primary or secondary stimulation, cells were harvested at the indicated time points, and total RNA was extracted using RNA ease Total RNA kit (Qiagen, Chatsworth, CA). Cytokine RNA levels were analyzed by RNase protection assay using RiboQuant multiprobe kit (PharMingen, San Diego, CA), following the instructions of the manufacturer. In brief, equal amounts of target RNA (2.5–9 µg) were hybridized overnight to a 32P-labeled RNA probe, which had been synthesized in vitro from a multicytokine template set (hCK3), after which, free probe and other single-stranded RNA were digested with RNase. The protected mRNAs were purified and resolved on a 6% denaturating polyacrylamide gel. Transcript levels were quantified by autoradiography and densitometric scanning of autoradiograms with exposures within the linear range. The cytokine transcripts were identified by the length of the respective fragments. RNA loading was standardized against the protected fragments of the housekeeping gene L32.
Detection of surface-associated lymphotoxin
Cells were stained by sequential incubation with mouse IgG1 (100 µg/ml), anti-LT polyclonal Ab (1 µg/ml (12), or control rabbit IgG, biotin-streptavidin-conjugated AffiPure mouse anti-rabbit IgG (heavy and light chain) (Jackson Immunoresearch Laboratories, West Grove, PA), and phycoerythrin-labeled streptavidin (Ancell, London, Canada). Stained cells were analyzed with a FACSort.
Detection of LT at the single cell level
T cells were stimulated with anti-CD3 mAb together with
CD32/B7.1 L transfectants for 48 h. Monensin (3 µM final
concentration; Hornby, Ontario, Canada) was added during the last
5 h. Cells were fixed with 2% paraformaldehyde, permeabilized
with PBS containing FCS (2%) and saponin (0.5%), and stained with
FITC-labeled anti-LT mAb (clone MTN1) (12) or FITC-labeled mouse
IgG1. Cells were washed and analyzed on a FACSort.
Statistical analysis
Paired t test was used to determine statistical significance of the data. Values of p < 0.05 were chosen for rejection of the null hypothesis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 (ranging from 60 to 150 ng/ml) together
with high levels of TNF- (Fig. 1
A). In
keeping with previous reports (7, 10), they also produced low levels of
IFN- and IL-13. These cytokines were produced with a different time
course in that maximal levels of IL-2 were observed after 24 h of
stimulation, as compared with 48 h for TNF- or IL-13 and
72 h for IFN- and LT3. The ability to produce LT3 was not
restricted to a subset of neonatal cells in that the large majority
(>90%) of anti-CD3/B7.1-activated CD4+ T cells
contained intracytoplasmic LT (Fig. 1B). Resting cells
did not contain intracytoplasmic LT, whereas 
70% of PMA plus
ionomycin-activated cells were positive (data not shown). Ag-specific
stimulation of neonatal CD4+ T cells, by means of
allogeneic dendritic cells, also induced high production of LT3.
Indeed, this cytokine was detected at similar levels in the culture
supernatants of primary MLR and of polyclonally restimulated primed
cells, in spite of the low frequency (<1%) of alloantigen-specific
cells in primary cultures (Fig. 1C). Although the large
majority of neonatal T lymphocytes are immunologically naive, they are
probably more immature than the naive T cells present in adult
individuals. Whereas these two types of naive cells express no or
little CD45RO Ag, the neonatal CD4+ T cells express less
CD45RA and CD31 but more CD38 than their adult counterparts (14, 15).
To determine whether the high production of LT3 by umbilical cord
blood T cells reflects their neonatal origin or their immunological
naivety, naive CD4+ T cells were isolated from adult blood
as CD4+CD45RO- CD31+ cells and
compared with memory cells (CD45RObright,
CD31-) for the production of LT3 as well as TNF- and
IFN-. As shown in Fig. 1D, adult naive cells
produced more LT3, similar levels of TNF-, and less IFN- than
their memory counterparts. Moreover, comparison of Fig. 1A
with Fig. 1D revealed that neonatal and adult naive cells
produced comparable levels of LT3 and TNF-, suggesting that the
cytokine production profile of umbilical cord blood CD4+ T
cells reflects their naivety, rather than their neonatal origin.
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, which is produced by a large variety of cells,
including nonlymphoid cells, the production of LT3 is restricted to
lymphocytes, mainly Th1 CD4+ T cells, CD8+ T
cells, and, to a lesser extent, NK cells and B cells (16). In the next
series of experiments, the production of LT3 by naive
CD4+ T cells was compared with that of: 1) Th1, Th0, and
Th2 effectors, 2) naive CD8+ T cells, and 3) B cells. In
Fig. 2, the production of LT3 and TNF- by naive T
cells was compared with that of effector cells derived from the same
naive precursors primed in the presence of exogenous IL-12 +
anti-IL-4 mAb (Th1 conditions), exogenous IL-4 (Th2 conditions), or
in culture medium alone (neutral conditions). The peaks of LT3 and
TNF- appeared earlier at restimulation than at priming and, as
expected (16, 17), they were higher in Th1 than Th2 effectors. However,
and most strikingly, following activation in neutral conditions, naive
T cells produced on average four times more LT3 and similar levels
of TNF- as Th1 effectors. Interestingly, whereas the priming
conditions markedly influenced the LT3- and
TNF--producing capacity of primed cells, they had only marginal
effects on the production of these cytokines in primary cultures. As
shown in Fig. 2, IL-12 had no effect, and IL-4 suppressed LT3 only
slightly.
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3 was not
restricted to the CD4+ subset. In five separate
experiments, highly purified CD8+ T cells were found to
produce 40.8 ± 5.6 ng/ml of LT3, as compared with
88 ± 16 ng/ml by CD4+ T cells isolated from the same
umbilical cord blood. In a next series of experiments, umbilical cord
blood B cells were activated with a variety of stimulants, including
PMA + ionomycin, soluble CD40L, Staphylococcus aureusCowan I bacteria, anti-µ and IL-4, used alone or in
combination, to determine optimal conditions for LT3 production. The
best inducer was PMA + ionomycin, which triggered the release of
2.3–5.2 ng of LT3 per 106 cells. Thus, naive
CD4+ T cells appeared to produce more LT3 than B cells,
recently differentiated CD4 Th1 effectors, and naive CD8+ T
cells.
The expression of high levels of LT and TNF- by naive T cells was
confirmed at the mRNA level. Cytokine mRNA was examined by RNase
protection assay before and after 5, 10, or 24 h of activation of
both naive and Th1 cells derived from the same cord blood samples. As
seen in Fig. 3, LT and TNF- mRNAs were expressed
later but at higher levels in naive than in Th1 cells. In particular,
the maximal level of LT transcripts observed after 24 h of
naive T cell activation was significantly higher than the peak value
observed after 10 h of Th1 cell activation
(p < 0.05).
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both as soluble homotrimers
(LT3) and as membrane-associated heterotrimers, made of LT and
LTß, a related type 2 integral membrane protein (reviewed in 12). Surface LTß (which predominantly exists as LT1ß2
complexes) and LT3 bind to different receptors and serve distinct
functions. LT3 binds to TNF-R1 and TNF-R2, with the same affinity as
TNF-, and to herpes virus entry molecule (HVEM), a recently
described member of the TNF-R family that is specifically expressed on
lymphocytes (12, 18). Surface LT1ß2 binds to LTß-R, a specific
receptor widely expressed on nonlymphoid cells including stromal cells
and follicular dendritic cells (FDC) (12). Therefore, it was of
interest to examine whether, in addition to producing LT3, naive
CD4+ T cells also expressed surface LT. As seen in Fig. 3
, A and D, LTß mRNA was constitutively
expressed on resting naive T cells, it was transiently down-regulated
during the first 10 h postactivation, and re-expressed at high
levels after 24 h. Th1 cells also displayed LTß mRNA before
stimulation, but, unlike in naive cells, these transcripts remained
down-regulated during the first 24 h of activation. Contrasting
with their constitutive expression of LTß mRNA, resting naive cells
did not display membrane-associated LT, which, however, became
progressively up-regulated during the first 3 days following
anti-CD3/B7.1 activation (Fig. 4). In contrast,
membrane LT was present on "resting" Th1 cells and slightly
up-regulated during the first 2 days of activation. Most interestingly,
the peak levels of membrane LT on Th1 cells were only slightly higher
than on naive cells. The presence of LTß on unstimulated Th1 cells
was in keeping with the recent finding that this heterotrimer is
constitutively expressed on Th1 as well as Th2 clones (19).
Alternatively, it might reflect the fact that the "resting" Th1
cells used in our experiments were examined after 4 days of culture in
IL-2-supplemented medium; indeed, IL-2 was shown to induce membrane LT
on resting primary T cells (19).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3, TNF-, IFN-, and IL-13. As already
mentioned, these cells were also reported to produce very low but
functionally sufficient amounts of IL-4 (2, 7). It is of note that,
regardless of their mode of activation, naive cells produce hardly
detectable levels of IL-10 (20–50 pg/ml) and no IL-5 (<50 pg/ml)
(Y.O., L-P.Y., and G.D., unpublished observations). The levels of
LT3 released by activated naive CD4+ T cells, of
neonatal or adult origin, are particularly impressive and appear to be
higher than those released by either naive CD8+ T cells,
adult memory CD4+ CD45RO+ T cells, Th1-like
effectors, or optimally activated B cells. When compared with Th1-like
cells generated after one cycle of priming in the presence of IL-12 and
anti-IL-4 mAb, naive CD4+ T cells produce 4 times
more LT, the same amounts of TNF-, but at least 10 times less
IFN-. Although we did not compare the production of LT by naive T
cells to that of fully differentiated Th1 cells, generated after
multiple cycles of stimulation in Th1-polarizing conditions, naive
CD4+ T cells may nevertheless be considered as major
producers of LT3.
The high expression of LT3, TNF-, and LTß during naive T
cell activation suggests an unsuspected role for these cytokines in the
early stage of T cell-dependent immune responses. These are initiated
in the T cell zones of secondary lymphoid organs where naive T cells
first encounter the Ag presented by interdigitating dendritic cells.
Within the first 3 days of priming, at the time when LT/TNF-
expression is maximal, some T cells accumulate in the outer T cell
zone, where they stimulate a vigorous early B cell response, whereas
others migrate to the germinal center (20). The intense wave of LT/TNF
production by naive T cells might contribute to the initial stage of
the immune response in a number of ways. For example, LT3, known to
be a potent B cell growth factor (21), may contribute to the early and
intense B cell proliferation occurring in the T cell area. Since
LTß signaling is required not only for the organogenesis of
secondary lymphoid organs (22, 23), but also for the maintenance of a
functional FDC network and germinal center response in the spleen of
adult mice (24), it is possible that naive T cell-associated LTß
may contribute to the germinal center response. This possibility is
challenged, but not excluded, by the recent finding that during
ontogenesis, B cell-associated but not T cell-associated LTß is
required for the development of normal splenic architecture, including
FDC networks (25, 26). Ectopic expression of LT gene induces the
local formation of "tertiary" lymphoid organs by mechanisms
involving both TNF-R1 signaling and increased expression of adhesion
molecules enhancing the local recruitment of lymphoid cells (27, 28).
Naive T cell-derived LT3 might play a similar role during primary
response and thus enhance the recruitment or the trapping of
lymphocytes in the secondary lymphoid organ where Ag is presented.
These cytokines might also regulate the fate of interdigitating
dendritic cells, whose survival is limited (29), and perhaps also
influence the development of naive T cells into Th1/Th2 effectors
(30).
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Acknowledgments |
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Footnotes |
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2 Y.O. and L-P.Y. contributed equally to this work.
3 Current address: Experimental Immunology Branch, National Institutes of Health, Bethesda, MD 20892.
4 Address correspondence and reprint requests to Dr. G. Delespesse, Centre Hospitalie Université de Montreal Research Centre, Notre-Dame Pavillon, Laboratory for Allergy Research (M4211-K), 1560 Sherbrooke Street East, Montreal, Quebec, H2L 4M1, Canada. E-mail address:
5 Abbreviations used in this paper: LT, lymphotoxin; h, human; FDC, follicular dendritic cells.
Received for publication November 30, 1998. Accepted for publication December 18, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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are ligands for herpesvirus entry mediator. Immunity 8:21.[Medline]
and ß revealed in lymphotoxin ß-deficient mice. Immunity 6:491.[Medline]
-dependent fashion. J. Exp. Med. 187:1009. 3 and membrane lymphotoxin- 1 ß 2 in lymphotoxin-induced inflammation: critical role of TNF receptor 1 signaling. J. Immunol. 160:485. and TNF- are required for IL-12-induced development of Th1 cells producing high levels of IFN- in BALB/c but not C57BL/6 mice. J. Immunol. 160:1708.This article has been cited by other articles:
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