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Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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A number of recent reports have revealed that both naive and memory T cells require continual interactions with MHC-expressing APCs in the periphery to survive (14, 15, 16, 17). In the case of naive T cells, this process demands that the peptides that are presented be the same as those that mediated positive selection in the thymus (18). An analogous situation for memory T cells would oblige the presented peptides to be identical with those encountered during the primary immune response; indeed, it has long been suggested (19) that intermittent stimulation by persisting Ag is necessary for the survival of both memory Th cells (5) and memory B cells (20). The only known means by which nonreplicating Ags can be retained in vivo for long periods is in the form of immune complexes on the surface of follicular dendritic cells (FDCs6; Ref. 21). If these are required to maintain Th cell memory, it follows that this memory should depend on the production of Abs by B cells. We tested this directly by examining the longevity of CD4+ T cell memory in mice that lack B cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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All animals were cared for in accordance with the U.K. Home
Office regulations and with the guidelines of the United Federation for
Animal Welfare. B6 µMT (22) B10 SCID, B6
CD11c-I-Ed (23), B6
I-A
-/- I-E+
(17), B6 Rag1-/-
(24), C57BL/6, B10.D2/n, B6xB10.D2/n
F1, and BALB/c mice were 6–10 wk old at the
start of each experiment. B6 µMT mice were backcrossed with congenic
B10.D2/n mice, and expression of H-2Kb and
H-2Dd on PBLs was assessed by FACS analysis using
biotinylated mAbs AF6-88 and 19-191 (both provided by A. Livingstone,
Center for Vaccine Biology and Immunology, University of Rochester
Medical Center, Rochester, NY), respectively, followed by
PE-streptavidin (Calbiochem, La Jolla, CA). B220 expression was
detected using FITC-labeled RA3-6B2 (PharMingen, San Diego, CA). Mice
were maintained on sterile food and bedding in filter-topped cages.
Generation of chimeric mice and immunization
Thymi were taken from C57BL/6 fetuses at day 14 of gestation and up to six were implanted under the kidney capsules of B10 SCID mice. Six to 10 wk later, reconstitution of T cells in peripheral blood leukocytes (PBL) was confirmed by FACS analysis using FITC-labeled anti-Thy1 (clone T24 obtained from American Type Culture Collection (ATCC), Manassas, VA). B10 SCID mice and B6 µMT mice were reconstituted i.v. with 107 B cells purified (25) from nonimmune C57BL/6 mice. At the same time, these mice were immunized i.p. with 50 µg soluble keyhole limpet hemocyanin (KLH; Calbiochem, San Diego, CA). KLH, possibly due to its large size, is one of the few Ags that is immunogenic in this form.
To prevent possible attack by NK cells, H-2b/d µMT mice were irradiated with 5 Gy 1 day before i.v. injection of 107 B cells from C57BL/6 or B10.D2/n mice. These mice were immunized as above 1 wk later to allow time for regeneration of T cells. Two weeks after immunization, all mice were bled, and serum Ig levels and anti-KLH IgG and IgM were measured as described (25). At the longest time point analyzed (12 wk), donor B cells were still detectable in splenocytes by FACS analysis of B220 expression (as above).
µMT mice were immunized i.p. with 50 µg soluble KLH, and 4 days later were injected i.v. with 50 µg (of Ag) soluble immune complexes; these were preformed in vitro by mixing monoclonal anti-DNP (clone DNPG1, IgG1 isotype) and DNP-KLH in a 3:1 ratio. Ten days after injection of immune complexes, serum anti-DNP and anti-KLH total Ig were measured.
B6xB10.D2/n F1 mice were irradiated with 10 Gy 1 day before i.v. injection of 107 bone marrow cells from B6 CD11c-I-Ed mice. These mice were immunized as above 8 wk later, by which time both T and B cells were present in PBL.
B6xB10.D2/n F1 mice were immunized i.p. with 50 µg KLH adsorbed to potassium alum (Sigma-Aldrich, Poole, U.K.). Six weeks later, splenocytes from these mice were depleted of dendritic cells (DCs; see below) and injected i.v. into unirradiated B6 Rag1-/- mice. These mice were injected i.v. with 5 µg soluble KLH on the following day. One week later, these mice were bled, and serum Ig levels and anti-KLH IgG and IgM were measured. BALB/c (H-2d) and C57BL/6 mice were immunized with 50 µg soluble KLH 7–9 wk before sacrifice and purification of DCs.
Limiting dilution analysis (LDA)
Frequencies of KLH-specific IL-2- and/or IL-4-producing T cells
were measured in reconstituted SCID and H-2b
µMT mice as described (26). All LDAs had
2 probabilities of >0.05, indicating that the
data was well fitted by a Poisson distribution of responder cells. To
measure the frequencies of H-2b- and
H-2d-restricted T cells in B cell-reconstituted
H-2b/d µMT mice and B and T cell-reconstituted
B6 Rag1-/- mice, and of
I-Ab- and I-Eb-restricted T
cells in B6 CD11c-I-Ed
B6xD10.D2
F1 bone marrow chimeras,
CD4+ T cells were highly purified from responder
splenocytes by depletion of MHC class II+,
CD8+, CD11c+, and
IgM+ cells using biotinylated mAbs M5/114 (ATCC),
53.6.72 (ATCC), and N418 (27), and goat anti-mouse IgM
(Southern Biotechnology Associates, Birmingham, AL), respectively,
followed by two rounds of MACS separation (Miltenyi Biotech, Bergisch
Gladbach, Germany). Purity of sorted cells was routinely >99.5%, and
these cells did not proliferate in response to either KLH or
immobilized anti-CD3 (145.2C11) unless exogenous APCs were added.
APCs used to enumerate generate I-Ab-
(equivalent to H-2b- in this case),
H-2d-, and I-Eb-restricted
responses were derived from C57BL/6, B10D2/n, and B6
I-A-/- I-E+ mice,
respectively. Otherwise, LDA was as above. It should be noted that T
cells restricted by hybrid
H-2b/H-2d MHC molecules
(e.g., I-Eb) will not be detected in the
comparisons between H-2b- vs
H-2d-restricted T cell responses.
Analysis of MHC restriction of Ag-specific T cells
Purified CD4+ T cells (2 x 106) from an H-2b/d µMT mouse given H-2d (C57BL/6) B cells were cultured with an equal number of irradiated (30 Gy) H-2d (B10.D2/n) APCs plus 100 µg/ml KLH and 100 U/ml IL-2. Two weeks later, cells were washed and recultured at 100 cells per well with 2 x 105 APCs (either H-2b or H-2d) and 100 U/ml IL-2, with and without KLH. After an additional 2 wk, wells were scored microscopically for cell growth. Numbers of positive wells/total wells: H-2b APC + KLH, 0/96; H-2b APC alone, 0/96; H-2d APC + KLH, 16/96; H-2d APC alone, 0/96 (p < 10-4, Fischer’s exact test).
Purification and depletion of DCs
DCs were purified from splenocytes as described
(28) and were additionally depleted of T cells using
anti-Thy-1 (T24)-coated dynabeads (Dynal, Oslo, Norway). In some
cases, 50 µg/ml soluble KLH was included during the overnight
culture. Their purity was ascertained by FACS analysis using
biotinylated anti-MHC class II followed by PE-streptavidin and
FITC-conjugated anti-CD11c Abs, and was typically 
80%. DCs were
depleted from total splenocytes by a single round of MACS separation
using biotinylated anti-CD11c. After depletion, contaminating
CD11c+ cells comprised <0.1%.
T cell stimulation by DCs
Graded numbers of purified DCs cultured either with or without KLH were incubated overnight with 5 x 104 E2-10HA cells, in 200 µl, in round bottom wells. The following day, 100 µl of supernatant was harvested from each well and IL-2 was measured (29).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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SCID mice have neither T nor B cells, but T cells can be
regenerated in these mice by implantation of fetal thymi from wild-type
mice. We immunized mice treated in this way with soluble KLH and
injected half of them at the same time with syngeneic B cells. Soluble
Ag was used to minimize the possibility of forming any artificial
deposits in vivo; thus, retention of Ag should occur solely by the
trapping of immune complexes on FDCs. The KLH preparation used in these
experiments is not mitogenic for B or T cells, elicits no response in
nude mice, and is dependent upon MHC class II presentation, as T cells
in class II knockout mice do not respond (B. McManus and D. Gray;
unpublished data). Our measure of memory in these experiments is the
frequency of responding T cells in LDAs, and so it is worth noting that
although this method may underestimate "memory" cells compared with
MHC tetramer staining, it does provide a readout of memory cell
function and a valid comparison of frequency between groups. The nature
of the assay ensures that we are measuring memory cells and not
effector cells, as the cultures required for the analysis extend over
at least 3 wk. Our studies indicate that effector cells will be picked
up in short-term but not in such long-term cultures. Two weeks after
immunization with KLH, all of the mice that had received B cells had
produced serum anti-KLH IgM and IgG Abs, indicating successful T
and B cell priming (data not shown). Ten weeks after immunization, we
measured the frequencies of KLH-specific Th cells in each mouse. At
this stage, the primary immune response has long since finished, and
the continued presence of elevated numbers of Ag-specific cells is
indicative of immunological memory. We found that those mice that had
received B cells had significantly higher numbers of KLH-specific Th
cells than those that had not (Fig. 1
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This demonstrates that CD4+ T cell memory is
impaired in the absence of B cells.
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, 2 wk after immunization there was
no difference in the level of T cell priming between mice that had, and
that did not have, B cells. This is in line with another report on T
cell priming in B cell-deficient mice (30). After 12 wk,
as with the SCID mice, the number of Ag-specific T cells had decayed
more rapidly in µMT mice that had not been given B cells (Fig. 2).
These data confirm that although B cells are not needed for the initial
expansion of Ag-specific T cells, they are vital for the maintenance of
Th cell memory.
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Ag presentation by B cells is not required for the maintenance of Th cell memory
Although FDCs can bind and retain immune complexes on their surface for long periods of time, they are unable to endocytose and process them for presentation to T cells (37, 38). For this, an intermediary APC is needed. Previous studies have shown that B cells are able to acquire Ag by endocytosis of bead-like, immune complex-coated bodies (iccosomes) derived from the dendrites of FDCs (37), and that they can present this Ag to CD4+ T cells in vitro (38, 39). Therefore, we investigated the requirement for Ag presentation by B cells to maintain Th cell memory. Because mice that lack B cells not only cannot form immune complexes, but also cannot support the development of FDCs (40, 41), we generated chimeric mice that contained B cells, in which half of the T cells could not recognize Ag presented by them. We backcrossed µMT mice (H-2b) onto the H-2d genetic background and generated F1 mice expressing both b and d alleles at the H-2 locus (H-2b/d µMT mice). These mice contain T cells that recognize Ag presented on either H-2b or H-2d MHC molecules. We then reconstituted these mice with H-2b B cells; the resulting chimeric animals generated serum Ab responses when immunized (data not shown), but only H-2b-restricted T cells could recognize Ag presented by B cells. We reconstituted a second group of mice in the reverse fashion, using H-2d B cells. All mice were then immunized as before, with soluble KLH. Two and 12 wk later, we performed LDAs of the CD4+ T cell from these mice, using either H-2b- or H-2d-expressing APCs. In this way, we were able to independently determine the frequencies of H-2b- and H-2d-restricted KLH-specific T cells.
Fig. 3 illustrates that 2 wk after
immunization, there was no difference in the level of T cell priming
between these two populations. This corroborates our earlier finding
that clonal expansion proceeds normally in the absence of B cells.
Twelve weeks after immunization, we found, to our surprise, that
Ag-specific T cells that could not recognize Ag presented by B cells
were still present at elevated levels, which were comparable to the
levels of Ag-specific T cells that could do so (Fig. 3). Thus, it seems
that although B cells are required for the survival of
CD4+ memory T cells (Figs. 1
and 2), they are not
needed as APCs (Fig. 3). It remained possible that the same T cell
could recognize KLH-derived peptides presented on both
H-2b and H-2d MHC
molecules. This is unlikely in view of the differences in structure and
in peptide-binding preferences (42) between
I-Ab and I-Ad molecules.
Nevertheless, we verified that this did not occur by stimulating bulk
cultures of CD4+ T cells from an
H-2b/d µMT mouse given
H-2d B cells, using
H-2d-expressing APCs. After 2 weeks, we purified
and restimulated these T cells under limiting dilution conditions using
either H-2d- or
H-2b-expressing APCs. Whereas clones of T cells
were readily stimulated by APCs expressing H-2d,
none were stimulated by APCs expressing H-2b (see
Materials and Methods). This confirms that
H-2d-restricted T cells in this mouse could not
recognize Ag presented on H-2b MHC molecules,
thereby strengthening the notion that CD4+ T cell
memory does not depend on Ag presentation by B cells.
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(43, 44).
As a result, no Ag retention on FDCs can take place in these mice.
µMT mice injected with immune complexes had Ag-specific serum Ab
levels after 2 wk comparable to those of µMT mice reconstituted with
B cells. However, the presence of specific Abs did not augment the
clonal expansion of Ag-specific Th cells after 2 wk; neither did it
prevent their disappearance by 12 wk after immunization (data not
shown). These data indicate that without the presence of B cells,
specific Ag-Ab complexes alone are insufficient to maintain Th cell
memory. Ag presentation by DCs is required for the maintenance of Th cell memory
Because Ag presentation by B cells is not needed, we wondered
whether Ag presentation by other cell types is required for the
survival of memory CD4+ T cells. Although FDCs
themselves bear MHC class II molecules on their surface, they do not
synthesize these molecules; rather, they are acquired from neighboring
B cells (38). Moreover, as mentioned earlier, FDCs are
unable to endocytose and process native Ag. Thus, in the above
experiments using H-2b/d µMT mice, the MHC
class II molecules on FDCs are of the same haplotype as those on the
donor B cells, indicating that direct recognition of Ag on the surface
of FDCs is also unnecessary for the survival of memory
CD4+ T cells. It has recently been shown that
noncognate recognition of MHC class II molecules on DCs can sustain
naive CD4+ T cells, which are otherwise
short-lived in vivo (17). Therefore, we examined the
ability of DCs to promote memory CD4+ T cell
survival. To this end, we generated mice in which half of the T cells
could recognize Ag in the periphery only when presented by DCs.
CD11c-I-Ed mice contain a transgene encoding the
-chain of I-Ed under the control of the
DC-specific CD11c promoter (23). As the transgene was
injected into eggs from C57BL/6 mice, in which the endogenous
I-Eb -chain is nonfunctional, surface
expression of I-E molecules (which are hybrid
I-Ed/I-Eßb, hereafter
termed I-Eb) is restricted to DCs
(23). To ensure positive selection of
I-Eb-restricted T cells, we reconstituted
lethally irradiated wild-type H-2b/d recipients
with bone marrow from these mice. This produced mice in which
I-Eb is expressed in the thymus and on DCs, but
not on B cells or other bone marrow-derived APCs.
I-Ab molecules are expressed in the same pattern
as in wild-type animals. After immunization with soluble KLH, all mice
produced specific serum Abs (data not shown).
As before, 2 and 12 wk later, we measured the frequencies of
KLH-specific CD4+ T cells restricted either by
I-Ab or by I-Eb. After 2
wk, we found that both I-Ab- and
I-Eb-restricted CD4+ T
cells had undergone clonal expansion; however, T cells restricted by
I-Ab were about three times more numerous than
those restricted by I-Eb (Fig. 4). This contrasts with the situation in
wild-type H-2b/d mice, in which both populations
of T cells expand equally following immunization (data not shown). This
may reflect the fact that the transgene-encoded
I-Ed is expressed on only 70% of DCs
(23). Nonetheless, 12 wk after immunization,
I-Eb-restricted KLH-specific Th cells (which can
recognize Ag only when presented by DCs) were still present at elevated
levels, no lower than were those of
I-Ab-restricted cells (which can recognize Ag
presented by all cell types). This illustrates that recognition of Ag
on DCs is sufficient to promote the survival of memory Th cells.
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), in accord with our earlier
experiments. Neither population increased in frequency during the first
week after transfer, although recipient mice did produce detectable
serum Abs (data not shown). At subsequent times, though, Ag-specific
H-2d-restricted CD4+ T
cells, which cannot recognize Ag presented by DCs, were rapidly lost,
whereas H-2b-restricted cells remained present at
high frequencies (Fig. 5). By 10 wk after transfer, Ag-specific T cells
restricted by H-2d were 
20-fold less numerous
than those restricted by H-2b. Similar decay in
H-2d-restricted memory has been observed in a
separate experiment in which the chimeras were immunized with a
different Ag, alum-precipitated OVA (data not shown). Presentation of
Ag by DCs, then, provides a vital survival signal for memory Th cells.
With this result "bystander" activation by cytokines, as reported
for CD8+ T cells (11, 12, 13), appears
to be ruled out as means of maintaining CD4+ T
cell memory, at least in response to a simple protein Ag.
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10
wk (data not shown), which agrees well with the values calculated from
µMT mice reconstituted with B cells (8.7 wk), and Rag1-knockout mice
reconstituted with wild-type splenocytes (10.2 wk). This half-life is
severely shortened in the absence of B cells or when Ag presentation by
DCs is prevented (2.1 and 2.3 wk, respectively). Tough and Sprent
(45) found that the intermitotic half-life of
"memory-phenotype" CD4+ T cells in
thymectomized mice is 1.7–1.9 wk, although they made no distinction
between true memory cells and recently activated effector cells.
Regardless, the closeness of these figures tempts one to speculate that
this is the intrinsic lifespan of a memory Th cell, and that within
this period such a cell must either be stimulated to divide or die of
neglect. Superficially, the results described in this paper appear to disagree with recent reports in which memory CD4+ T cells survive for long periods in the absence of Ag and even MHC class II (46, 47). Both of these studies performed adoptive transfer of TCR transgenic T cells into mice that were deficient of T cells (RAG-1 or MHC class II knockouts). In normal mice in the face of emigration from the thymus and of Ag-driven expansion of multiple T cell clones in a polyclonal environment, a homeostatic mechanism must act very stringently to limit clone size and memory cell lifespan. In lymphopenic mice, competition for "niches" is less intense, and homeostasis may well be relaxed. An illustration of the changes that can occur in T cell physiology in lymphopenic, mice was given by recent studies; in the periphery of lymphopenic, but not intact hosts, T cell proliferation was driven by the low affinity peptide ligands (18, 48). Our data suggest that memory CD4+ T cells are not exempt from selection pressures, and that under normal circumstances intermittent Ag stimulation can protect memory cells from homeostatic deletion. Whether this is an absolute Ag dependence is not addressed here, but we suggest that the period of influence of Ag on CD4+ memory cells is a long one. Swain et al. (47) may be correct in stating that excessive antigenic stimulation during a primary response can impair the efficient development of memory T cells (leading to clonal exhaustion?); however, their suggestion that absence of antigenic stimulation enhances survival is not borne out by the experience of vaccination, where several boosts are required for long-term protection.
Long-term Ag presentation by DCs in vivo
If Ag presentation by DCs is needed for the survival of memory Th
cells, it follows that in immune animals there must be DCs actively
presenting Ag long after immunization. Therefore, we sought to detect
these cells directly ex vivo. We immunized mice with KLH, and 7–9 wk
later purified DCs from these and naive controls, in the presence or
absence of exogenous KLH. We then cultured these cells in vitro with
the KLH-specific T cell hybridoma E2-10HA (49). The next
day, we measured the IL-2 production by the T cell hybridoma, as an
indirect measure of presentation of KLH by the DCs. Fig. 6A shows that DCs from both
naive and immune mice were able to stimulate the T cell hybridoma when
provided with exogenous KLH. However, only DCs isolated from immune
mice were able to elicit a response in the absence of added Ag,
demonstrating that at least some of these cells bear MHC class II
molecules presenting KLH peptides.
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B, DCs from immune
H-2d, but not H-2b mice
were able to stimulate the T cell hybridoma, as expected. When DCs were
purified from a mixed population of cells from immune
H-2b and naive H-2d mice,
no response was seen, testifying that Ag is not acquired during the in
vitro purification procedure. The same
H-2d-expressing DCs generated a strong response
when exogenous KLH was added to the cultures (Fig. 6B).
These data imply that DCs are able to present Ag in vivo long after
that Ag is originally encountered. Are these long-lived DCs that encountered Ag during the original immune response, or are they newly formed cells that have recently acquired Ag? We favor the latter idea. Recent experiments by Garcia et al. (46), in which purified DCs were injected into Rag1-/- recipients, suggest that mature DCs do not survive for longer than 3 wk in vivo. It is possible that some DC precursors may be able to capture Ag and wait for a considerable time before differentiating and presenting it to memory CD4+ T cells. However, this too seems unlikely as DC precursors have been shown to rapidly mature in response to tissue injury (50) or to inflammatory stimuli (51, 52). There are a number of possible mechanisms by which newly formed DCs could acquire Ag and present it to memory CD4+ T cells. FDCs, on which long-term Ag retention takes place (21), are located in B cell follicles, and a small population of DCs has lately also been shown to be present at this site (53, 54). The simplest scenario, then, is that memory CD4+ T cells periodically traffic through B cell follicles, and there they encounter specific Ag presented by resident DCs. In accordance with this, most T cells found in B cell follicles are CD4+ and express ‘memory-phenotype’ surface markers (55, 56, 57). It is conceivable, though, that DCs obtain Ag from FDCs and subsequently migrate out of the B cell follicle into the T cell zone, where there is a much larger audience of T cells. It is also unclear whether DCs can directly acquire Ag from FDCs or whether an intermediary cell is involved. Both B cells (37, 38, 39) and macrophages (37) are able to endocytose FDC-derived iccosomes in vivo, but this process has so far not been demonstrated for DCs (possibly because they have only recently been found in B cell follicles). In contrast, Townsend and Goodnow (58) have demonstrated extremely efficient transfer of Ag from B cells to other (unidentified) APCs, and subsequent presentation to CD4+ T cells in vivo.
Concluding remarks
We have shown that CD4+ T cell memory decays in the absence of B cells. This is markedly different from the published reports on CD8+ T cell memory (59, 60), implying that CD4+ and CD8+ memory T cells are not maintained by the same mechanism. Notably, Ag presentation by B cells is not required for their prolongation of CD4+ T cell memory. This argues that they may act by enabling the development of FDCs, and by the formation of immune complexes, together allowing long-term Ag storage. Although the exact role of B cells is not clarified, the results do stress the importance of including B cell, as well as T cell epitopes when designing novel vaccines. We have also found that Ag presentation by DCs takes place long after immunization, and that this is essential for the survival of memory CD4+ T cells. The exact means by which DCs obtain Ag, and the location of their interaction with memory CD4+ T cells, remain to be elucidated.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dominic van Essen at the current address: Basel Institute for Immunology, Grenzacherstrasse 487, Postfach, Basel 4005, Switzerland.
3 Current address: Division of Genetic and Molecular Medicine and Sheffield Institute for Vaccine Studies, University of Sheffield Medical School, Sheffield S10 2RX U.K.
4 Current address: Institute fur Immunologie, University of Munich, Germany.
5 Current address: Institute of Cell, Animal, and Population Biology, University of Edinburgh, Ashworth Laboratories, King’s Buildings, West Mains Road, Edinburgh EH9 3JT U.K.
6 Abbreviations used in this paper: FDC, follicular dendritic cell; PBL, peripheral blood leukocytes; KLH, keyhole limpet hemocyanin; ATCC, American Type Culture Collection; LDA, limiting dilution analysis; DC, dendritic cell.
Received for publication May 3, 2000. Accepted for publication July 13, 2000.
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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). The
remainder did not receive any B cells (
). Each point represents the
frequency in one mouse. Geometric mean frequencies (lines): SCID + T
cells and B cells, 1.8 x 10-4; SCID + T cells only,
1.2 x 10-5; p = 0.012,
two-tailed, unpaired Student’s t test. Unimmunized mice
typically exhibit frequencies of <2 x 10-6 (data
not shown). This result is representative of three similar
experiments.
). B, Ag presented by ex vivo DCs is acquired in
vivo. Presentation of KLH peptides by DCs purified from BALB/c
(first row) or C57BL/6 (second row) mice
9 wk after immunization with KLH, purified from a mixture of
splenocytes from unimmunized BALB/c and immunized C57BL/6 mice
(third row), or purified from unimmunized BALB/c mice
and pulsed with KLH (fourth row). The bottom row shows
the IL-2 production by the KLH-specific T cell hybridoma in the absence
of any DCs. The data are representative of three separate
experiments.