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Cell Clones Can Be Defined Phenotypically and Functionally as Th1/Th2 Cells and Illustrate the Association of CD4 with Th2 Differentiation1
*
Department of Biology and
Section of Immunobiology, Yale University, New Haven, CT 06511;
Imperial Cancer Research Fund Laboratories, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ß T cells into Th1 and
Th2 subsets has become an established and important paradigm. The
respective activities of these subsets appear to have profound effects
on the course of infectious and autoimmune diseases. It is believed
that specific programs of differentiation induce the commitment of an
uncommitted Th0 precursor cell to Th1 or Th2. A component of these
programs is hypothesized to be the nature of MHC-peptide antigen
presentation to the ß T cell. It has heretofore remained uncertain
whether a Th1/Th2 classification likewise defines, at the clonal level,
T cells. Such cells do not, as a general rule, express either
CD4 or CD8ß, and they do not commonly recognize peptide-MHC. In
this report, cell clones are described that conform strikingly
to the Th1/Th2 classification, both by cytokine expression and by
functional activities of the clones in vitro and in vivo.
Provocatively, both the cell clones and primary cells in
vivo showed a strong association of the Th2 phenotype with CD4
expression. These results are discussed with regard to the
immunoregulatory role that is increasingly emerging for cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß and T
cells (1, 2). These two lineages of T cells share some common
characteristics, such as the association of the TCR chains with CD3
molecules (3), the expression of certain other surface molecules (4),
and cell functions, such as cytotoxicity (5, 6, 7). However, they differ
significantly in their anatomical distribution, ontogeny, and
immunobiologic roles (3, 8, 9, 10, 11, 12, 13, 14, 15).
Over the past few years, a classification of CD4+
ß T cells has been established, primarily according to the cells’
pattern of cytokine production and consequent physiologic function
(16). Th1 cells produce IFN- and IL-2, which activate T cells and
macrophages to attack intracellular pathogens and promote, through B
cell help, the synthesis of particular Ig isotypes (e.g., murine
IgG2a). Th2 cells, conversely, produce IL-4, IL-5, and IL-10,
which help B cells synthesize other Ig isotypes (e.g., murine
IgG1 and IgE) commonly associated with the attack on extracellular
pathogens. The analysis of numerous infection systems and autoimmune
diseases indicates that the skewing of the response to either Th1 or
Th2 activation, respectively, has significant consequences for
clearance of pathogen and/or characteristics of lymphoid infiltration
(17, 18, 19, 20). In addition, Th2 cells may prove to be major physiologic
down-regulators of Th1 responses (21).
How T cells might fit into the Th1/Th2 pattern has not been
clearly elucidated. First, most T cells are not
CD4+; thus, if CD4 expression were an important component
of Th1/Th2 differentiation, one might expect that T cells
would not conform to this paradigm. Furthermore, several experiments
using mice congenitally deficient in the synthesis of ß T cells
have demonstrated that cells, unlike ß T cells, are either
incapable of or at best inefficient in providing
Ag-specific responses of the kind responsible for pathogen clearance or
for Ag-specific autoimmunity (14, 15, 22, 23, 24).
This notwithstanding, human and murine cells have been
demonstrated to provide B cell help (25, 26, 27, 28, 29, 30, 31) and, in association with
this, were clearly shown to produce IL-4 (26, 29), a signatory Th2-type
cytokine. Likewise, mice and humans infected with bacteria, viruses, or
protozoa have demonstrated increases in lymphoid or intraepithelial
cells, suggesting an involvement of cells in the nature
of the host response (32, 33, 34, 35, 36, 37, 38, 39). Indeed, when the intracellular
expression of cytokines by such responding cells was examined,
it revealed Th1- and Th2-type patterns that paralleled the prevailing
Th1 and Th2 ß T cell responses (40). Since then, additional
studies have demonstrated the production of Th1 and Th2 cytokines by
populations of cells (41). These data have provoked the question
of how closely the production of cytokines by cells might
conform at the clonal level to the Th1/Th2 paradigm defined for ß
cells.
In this study, cellular, molecular, and functional evidence is
provided for the classification of T cell clones as Th1 or Th2.
We discuss the potential relevance of this to emerging bioassays for
cells (15, 24, 42, 43, 44, 45) and to the idea that Th1/Th2
determination results from the mode of presentation of peptide-MHC
by a professional APC to a responding CD4+ T cell (46, 47).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice were bred and maintained in specific pathogen-free animal
facilities at Yale University (New Haven, CT).
TCR-/- mice (H-2b/d) were generated
by gene targeting (48). TCRßx-/- mice were
generated locally by breeding TCRß-/- mice
(H-2b) (49) with TCR-/- (H-2b)
mice (50). CB17.SCID mice (H-2d) were obtained from The
Jackson Laboratory (Bar Harbor, ME).
Establishment of cell lines
TCR-/- splenocytes (2 x
106/ml) were cultured in Click’s medium plus 5 U/ml IL-2
(supernatant of EL4), 5% heat-inactivated FCS (HyClone, Logan, UT),
and antibiotics (Life Technologies, Grand Island, NY). The cultures
were replenished with medium every 3 to 4 days. Irradiated (3000 rad)
feeder cells (BALB/c splenocytes, 106/ml) were added 2 wk
after the initial culture and weekly thereafter for 4 wk. Once a line
was established, it was weaned off feeders.
Establishment of cell clones
Limiting dilution of cell lines was performed on
irradiated feeder cells in 96-well microtiter plates at
n 
1 cell/well. Medium was replenished every 3 to 4
days, and irradiated (3000 rad) feeder cells were provided weekly for
the first few weeks and at 2-wk intervals subsequently until the
establishment of clones, after which the supply of feeder cells was
gradually stopped. All experiments presented in this study were
performed with established clones (free of APC).
Monoclonal Abs
The following directly conjugated mAbs were purchased from
PharMingen (San Diego, CA): PE-conjugated
anti-CD3 (2C11), anti-TCR (GL3), and anti-CD8
(53-6.7); FITC-conjugated anti-TCRß (H57) and anti-CD4
(RM4-5); and biotin-conjugated anti-Thy1.2 (53-2.1), anti-CD45R
(B220, RA3-6B2), and anti-CD40 ligand (CD40L, MRL). Hybridoma
culture supernatants were either maintained in this laboratory (2C11,
H57, and GL3) or provided by Dr. C. Janeway, Jr. (Howard Hughes Medical
Institute, Yale University: 2.4G2, anti-Fc receptor; 212A.1,
anti-I-Ab/d), or Dr. Albert Bendelac (Princeton
University, Princeton, NJ; anti-CD1).
Cell staining and FACS analysis, sorting, and activation
Single cell suspensions (
106 cells/ml) were
incubated with PE- or FITC-conjugated Abs at pretitrated dilutions on
ice for 30 min, followed by washing three times with PBS-1% FCS and
0.02% sodium azide. Biotin-conjugated mAbs were further incubated with
fluorescence-conjugated streptavidin. Stained cells were fixed in
PBS-1% paraformaldehyde and analyzed on a FACScan (Becton Dickinson,
Mountain View, CA). Dead cells and nonlymphoid cells were excluded by
selective gating on forward and side scatter. For sorting, splenocytes
(108/ml) were stained with PE-conjugated anti-TCR
(GL3) and FITC-conjugated anti-CD4 (RM4-5). After washing, cells
were resuspended at 2 x 107/ml in PBS-2% FCS for
sorting on a FACStar (Becton Dickinson), after which they were either
used directly as a source of RNA (ex vivo sample) or activated in
Click’s medium, 5% FCS, and 2.5 µg/ml Con A for 48 h before
being used as a source of RNA (activated sample). In the latter case,
the viability of cells was confirmed before and after harvest by trypan
blue exclusion.
RT-PCR for cytokine, Fas, and Fas ligand (FasL) mRNA
RNA was prepared from cell clones (2.5 x
106 cells) by RNAzol (Biotecx Laboratories, Inc.,
Houston, TX) and reverse transcribed into single strand cDNA using an
oligo(dT) primer (Pharmacia, Piscataway, NJ) and Moloney murine
leukemia virus reverse transcriptase (Life Technologies) at 37°C
(neutral pH) for 60 min. Two microliters of the cDNA (100 µl) was
amplified with primers specific for IL-4, IL-5, IL-10, IFN-,
TGF-ß, Fas, and FasL together with hypoxanthine phoshoribosyl
transferase (HPRT; as a control) in the presence of 100 ng of the 5'
and 3' primers, 1 µl of dNTPs (10 mM), 1.5 mM MgCl2, and
1 U of Taq polymerase (Boehringer Mannheim, Indianapolis, IN). The PCR
reactions were denatured at 94°C for 3 min followed by 35 cycles of
94°C for 20 s, 60°C for 20 s, and 72°C for 40 s
and a final extension at 72°C for 7 min. PCR products were analyzed
on 1.5% agarose gels. Primers were synthesized in the Keck Facility of
Yale University, and the primer sequences for cytokines were adopted
from the report by Reiner et al. (51) with corrections: IL-4,
5'-CATCGGCATTTTGAACGAGGTCA-3' and
5'-CTTATCGATGAATCCAGGCATCG-3'; IL-5,
5'-GAAAGAGACCTTGACACAGCTG-3' and
5'-GAACTCTTGCAGGTAATCCAGG-3'; IL-10,
5'-CCAGTTTTACCTGGTAGAAGTGATG-3' and
5'-TGTCTAGGTCCTGGAGTCCAGCAGACTCAA-3'; IFN-,
5'-CATTGAAAGCCTAGAAAGTCTG-3' and 5'-CTCATGAATGCATCCTTTTTCG-3';
HPRT, 5'-GTTGGATACAGGCCAGACTTTGTTG-3' and
5'-GAGGGTAGGCTGGCCTATGGCT-3'; and additionally, Fas,
5'-ATCCGAGCTCTGAGGAGGCGGGTTCATGAAAC-3' and
5'-GGAGGTTCTAGATTCAGGGTCATCCTG-3'; and FasL,
5'-CAGCTCTTCCACCTGCAGAAGG-3' and
5'-AGATTCCTCAAAATTGATCAGAGAGAG-3'.
Quantitative RT-PCR
Cytokine mRNA was quantitated by competitive PCR (51), employing the simultaneous amplification by the same primers of known quantities of competitor DNA fragments. The competitor (provided by Dr. Richard Locksley, University of California, San Francisco, CA) differs from the cDNA of interest by an insert that allows the relative amounts of the two amplification products to be distinguished by gel electrophoresis.
To establish comparable amounts of cDNA template for subsequent
analysis of cytokine gene expression, the cDNA was used as a template
for the amplification of the HPRT housekeeping gene in the presence of
varying amounts of HPRT competitor fragment (see Fig. 6
B and
accompanying text in Results; numbers at the top
of the lanes refer to fold dilutions of the competitor HPRT fragment
used). At each dilution of competitor, the ratio of the product derived
from the cellular cDNA to the product derived from the competitor was
assessed on a Bio-Rad Laboratory Densitometer (Richmond, CA), using
photographic negatives from ethidium-stained agarose gels. These ratios
were plotted against competitor concentration, and the linear ranges
established and compared by determination of regression (Fig. 6, C and D, and accompanying text in
Results). This allowed us to compare the competitor
concentrations required to obtain specific ratios of cell product to
competitor product for each of the cDNAs, from which we could determine
the relative operational concentrations of the cDNAs under study.
Dilutions of those equivalent concentrations of cDNAs were then used as
substrates for amplification of cytokine genes in the presence of a
range of competitor concentrations. The ratios of cDNA product to
competitor product were likewise plotted against the competitor
concentration. When these plots were compared, it was possible to
assess the relative abundance of cytokine cDNA in the different samples
(see Fig. 6, E–I, in Results).
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T cell
clones
TCR and gene rearrangements of clones were
independently amplified by RT-PCR using "hot start" (denaturing at
94°C for 5 min) followed by 38 cycles of 94°C for 1 min, 58°C for
40 s, and 72°C for 1 min in a DNA thermal cycler 480
(Perkin-Elmer/Cetus, Emeryville, CA). The amplified products were
purified (Qiagen, Chatsworth, CA), ligated, and transformed into
Escherichia coli using the TA method (Invitrogen, San Diego,
CA). Sequencing analysis was performed using Sequenase (52),
[35S]dATP, and SP6-, T7-, or TCR-specific
oligonucleotides as primers, as previously described (28).
Adoptive cell transfer and cell tracing
Splenocytes (10–15 x 106) from
TCRßx-/- mice (10–16 wk old) mixed with 3 to
5 x 106 cloned cells were injected i.v. into
CB17.SCID mice (6–8 wk-old). Cloned cells (3–5 x
106) were also i.v. transferred to
TCRßx-/- mice. For cell tracing, clones and
splenocytes from a TCRßx-/- mouse were labeled with
the fluorescent dye DiI (Molecular Probes, Inc., Eugene, OR) at 37°C
for 30 min before adoptive transfer. Transferred cells that were
labeled could be observed in frozen spleen sections by fluorescence
microscopy, as previously described (27).
ELISA quantitation of Ig and cytokine levels
For Ig quantitation, recipients were bled, and individual serum
samples were collected every 2 wk postreconstitution. Total levels of
serum IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA were determined by ELISA
as described previously (26, 27), using reagents from Southern
Biotechnology Associates, Inc. (Birmingham, AL). Briefly, microtiter
ELISA plates (Dynatech Laboratory, Inc., Chantilly, VA) were coated
with goat anti-mouse IgH+L (5 µg/ml) in coating buffer (carbonate
buffer, pH 9.6). After blocking the plates with 1% BSA (PBS containing
1% BSA), diluted serum samples (1/100 in blocking buffer) were added
in duplicate. For standards, serial dilutions of mouse IgM, IgG1,
IgG2a, IgG2b, IgG3, and IgA (Southern Biotechnology Associates) were
also added in duplicate, starting at 1 µg/ml. After incubation and
washing, alkaline phosphatase-conjugated goat anti-mouse IgM, IgG1,
IgG2a, IgG2b, IgG3, and IgA were added individually. The enzymatic
reaction was developed by adding substrate p-nitrophenyl
phosphate and was stopped by adding 1 N NaOH. The plates were read at
an absorbance of 405 nm on an microplate reader (Dynatech). The
concentrations of Ig isotypes were determined by referring to standard
curves performed in the same assay with known concentrations of various
mouse Ig isotypes using the equation y = intercept
+ slope x log(x); the actual serum
concentrations were obtained by y x the serum dilution
(i.e., 100). Note that the concentrations quoted in the figures in this
paper are for the sera and are calculated from the concentrations of
serum dilutions that were experimentally determined and compared with
concentrations of standards in the same ranges as the serum dilutions.
Secreted cytokines (IL-4 and IFN-) in culture supernatants were also
measured by ELISA using mAbs against murine IL-4 or IFN-
(PharMingen) together with different concentrations of rIFN- and
IL-4 (Life Technologies) as standards. The concentrations of IL-4 and
IFN- in the culture supernatants were converted as described for Ig
isotypes.
Detection of germinal centers (GC)
GC formation was examined in the spleens of reconstituted SCID
mice and TCRßx-/- mice, respectively, using
immunohistochemistry as reported previously (27, 28).
Histologic examination
Liver, kidney, intestine, and lung from the reconstituted SCID
or TCRßx-/- mice were fixed in 10% buffered
formalin, paraffin embedded, and stained with hematoxylin and eosin.
The sections were examined microscopically for lymphocytic infiltration
to evaluate the presence of graft-vs-host-disease.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cell clones derived
from TCR-/- mice
Two cell lines were derived from splenocytes of two
TCR-/- mice (H-2b). They were
CD4+ and CD4-CD8- (double
negative, DN), respectively. From these, a total of five cell
clones were obtained by limiting dilution. Two (G5 and H4) were
CD4+; three were CD4-,CD8- (A3,
F6, and H2; Fig. 1A).
Also studied was the expression on the clones of adhesion molecules
(ICAM-1, LFA-1, and lymphocyte Peyers patch adhesion molecule-1
(LPAM)), costimulatory molecules (CD28 and CD40 ligand), and CD1 that
are either known or speculated to play important roles in T cell
function. Examples of the range of expression levels are provided in
Figure 1B, and the data are summarized in Table I. All five cell clones expressed
LFA-1, ICAM-1, and CD28 similarly, while the expressions of CD40L and
4 integrin (LPAM) were more heterogeneous. The data are
consistent with previous studies of cell populations (4, 53, 54), in that expression of CD28 is more variable than is generally the
case for ß cells. Interestingly, CD1 was clearly expressed by all
the clones in which it was tested, consistent with the recent report
that CD1 is expressed by various hemopoietic cells (55).
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cell clones
Cytokine mRNA expression by clones was determined by
RT-PCR. Both CD4+ T cell clones (H4 and G5)
conformed strikingly to a typical Th2 phenotype: high levels of IL-4,
IL-5, and IL-10 and undetectable levels of IFN- (Fig. 2A) or IL-2 (data not
shown). By contrast, all DN T cell clones conformed to a typical
Th1 phenotype, displaying high levels of IFN- (Fig. 2A) and IL-2, against undetectable IL-4, IL-5, and
IL-10 (Fig. 2A).
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ß T cells expressing high ratios of FasL:fas, and
Th2 CD4+ ß T cells expressing higher ratios of
fas:FasL (Th0 cells are reported to express Fas and FasL approximately
equivalently) (57). To test the degree to which the clones
conformed to this Th1/Th2 dichotomy, RT-PCR was again applied. D10, a
CD4+ ß+ Th2 clone, and B3B3, a
CD4+, ß+ Th0 clone were included as
positive controls. The data (Fig. 2
B) show that the
FasL:Fas expression ratios of the cell clones conformed to their
classification as Th2 and Th1 cells, respectively: G5 and H4 (Th2)
expressed higher levels of Fas than of FasL, while A3, F6, and H2
(together with an additional IFN--expressing clone, A7)
showed significant levels of FasL, but negligible expression of Fas
(Fig. 2B).
To examine whether gene expression patterns by the clones were
representative of effector molecule production, the secretion of IL-4
and IFN- was examined by ELISA. Consistent with the RT-PCR data,
secreted IL-4 was detected only in the supernatants of H4 and G5, while
secreted IFN- was detected only in the supernatants of F6 and H2
(Fig. 2C) and A3 (data not shown).
Regulatory role of cells in class switching of B cells in
vitro
A series of experiments was undertaken to determine whether the
functional capabilities of the clones likewise conformed to the Th1/Th2
classification. First, the clones were activated in vitro with
anti-CD3 and cocultured with naive, primary B cells derived from
TCRßx-/- mice; such cells were uninfluenced by
any prior exposure to T cells. Igs of different isotypes were measured
in the culture supernatants (n = 3) harvested 7
days postincubation. All + clones elicited Ab
secretion (Fig. 3), but in the absence of
stimulation by anti-CD3, most of the Ig produced by B cells was
IgM. Conversely, activation of all the + clones
followed by coculture with naive B cells provoked IgG secretion.
However, the putative Th2-+ clones, G5 and H4,
induced class switching primarily to IgG1, whereas the putative Th1
+ clones induced class switching primarily to IgG2a.
This conforms strikingly to the behavior of Th1 and Th2 ß T cell
clones (Fig. 3).
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To test whether the Th1/Th2 classification applied to the
+ clones in vivo, CB17.SCID recipients
(n = 3–4/group) were adoptively transferred
with various T cell clones, admixed with splenic B cells derived
from TCRßx-/- mice. As controls, clones
alone or TCRßx-/- splenic B cells alone were also
transferred to CB17.SCID recipients (n =
2–3/group). The engraftment of cells was confirmed in the short term
by tracing, using fluorescent dye (DiI)-labeled clones or B
cells (as in our previous studies (27)) and in the longer term by FACS
analysis at 4 wk postadoptive transfer (Fig. 4A).
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T cell clones together with splenic B cells
from TCRßx-/- mice. As shown in Figure 4B, the DN (Th1-like) T cell clone, F6, induced GC
formation by B cells from TCRßx-/- mice. GC
reconstitution was previously shown using the Th2 clone, G5 (27). No
GCs formed when splenocytes from TCRßx-/- mice were
inoculated without clones (Fig. 4C) (27). To
extend this finding, DN and CD4+ clones were
transferred directly to TCRßx-/- mice, in which, in
the congenital absence of T cells, most Ab production is IgM and in
which GCs do not develop (28). Again, GC formation was induced by both
putative Th1 and putative Th2 clones (data not shown).
Consistent with our earlier findings (28), no GCs were observed in the
TCRßx-/- recipients that received PBS
only.
At various time points post-transfer, the CB17.SCID recipients were
also assessed for serum Ig. The data (Fig. 5) reveal that all +
clones sustained the production of IgG by transferred B cells, albeit
at low levels. Conversely, Abs in SCID mice receiving B cells alone
were almost exclusively IgM. Strikingly, IgG1 production was
reproducibly higher in mice receiving putative Th2 clones G5 and H4,
whereas IgG2a production was higher in mice receiving putative Th1
T cell clones, e.g. F6 (Fig. 5).
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T cell
clones
The TCR/ gene usage of the clones was defined by RT-PCR,
cloning, and sequencing (Table II). Both
CD4+ Th2-type + T cell clones used
V7-J1 gene segments, whereas the DN Th1-type +
T cell clones used V1-J4 gene segments. All the
+ T cell clones used in this study expressed
V6-J1 gene segments (Table II). The two CD4+ Th2-type
T cell clones that were originally derived from the same line
shared identical joining sequences for both V and V gene
segments, indicating that they are in all likelihood sister subclones
of a single progenitor. The junctional sequences in two DN Th1-type
T cell clones (A3 and H2) were likewise identical. Thus, the
stable retention of a uniform Th1 or Th2 phenotype by sibling clones
over time (the clones have been maintained for >2 yr), which is a
further criterion of Th1/Th2 differentiation in ß cells, is
likewise a characteristic of the + clones. A third
clone, F6 (DN, Th1 type), expressed different V1-J4/V6-J1
rearrangements (Table II). Strikingly, the V1-J4 rearrangement of
this clone is identical with a monoclonal rearrangement (15.32) (28)
microdissected from a GC of a different, infected
TCR-/- mouse in which there was marked IgG
expression.
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cells in vivo
The Th2 clones described here are CD4+. Compared
with DN and CD8 + cells, CD4+
cells are rare, even in TCR-/- mice (48, 60). It therefore
seemed surprising that the exercise of cloning Th2 +
clones yielded cells that were CD4+. In turn, this provoked
the hypothesis that CD4 expression might be more strongly associated
with Th2 differentiation than with Th1 differentiation. To examine this
in vivo, an experiment was undertaken to compare the amount of IL-4 RNA
expressed by CD4+ cells and CD4-
cells. CD3+ TCR+ cells were isolated
directly from TCR-/- spleens and sorted by FACS into
CD4- or CD4+ subsets (windows R2 and R3 in
Fig. 6A). In one
experiment, the sorted CD4+ + cells and
CD4- cells were used immediately ex vivo to prepare
RNA. In a second experiment, the CD4+ cells and the
CD4- cells were each activated for 48 h in the
presence of Con A (2.5 µg/ml) and then harvested for RNA. In each
case, the extracted RNA was used as substrate for cDNA synthesis. To
establish comparable levels of cDNA template for subsequent analysis of
cytokine gene expression, the protocol described in Materials and
Methods was applied. Briefly, cDNAs were compared for their
capacity to act as templates for HPRT gene amplification in the
presence of varying amounts of competitor HPRT fragment. Data for the
activated CD4+ + and CD4-
+ samples are shown in Figure 6B (numbers
at the top of the lanes refer to fold dilutions of the
competitor HPRT fragment used). At each dilution of competitor,
densitometry was used to determine the ratio of the product derived
from the cellular cDNA to the product derived from the competitor;
these ratios were then plotted against the competitor concentration,
and the linear range was established (Fig. 6, C and
D; equations for linear regression: activated
CD4+ cells, y = -2.9583e-2 +
29.305x, R2 = 0.977; activated
CD4- cells, y = 0.19125 +
30.626x, R2 = 0.983). From these
plots, the relative operational concentrations of the cDNAs could be
determined, allowing equal amounts of cell cDNA to be used for
subsequent competitive amplification of cytokine genes. When this was
attempted for IL-4, using a range of concentrations of competitor, it
was immediately apparent that cDNA from the activated CD4+
cells competed much more effectively for the IL-4 primers than did cDNA
from CD4- cells (Fig. 6E), in support of
the stated hypothesis. The same was true for the nonactivated ex vivo
samples (Fig. 6G). To calculate more precisely the
difference in relative IL-4 cDNA concentrations, a broad range of
competitor was used with each cDNA sample (Fig. 6F)
to establish the range (in each case) over which the ratio of product
derived from the cDNA compared with product derived from the competitor
showed a linear relationship to input competitor (Fig. 6, H
and I show the data for the activated CD4+ and
CD4- samples). Comparison of these plots revealed IL-4
expression in activated CD4+ cells to be 17.4 times
more abundant than IL-4 expression in activated CD4-
cells (see Fig. 6). When the same approach was applied to the ex
vivo samples, a similar excess of >10-fold IL-4 RNA was found in the
CD4+ + sample (Fig. 6G;
quantitation data not shown). The expression of IL-10 showed a similar
pattern. These data demonstrate that the expression of Th2 cytokines is
at least an order of magnitude greater in peripheral CD4+
+ cells than in DN + cells. This
did not apply to IFN-, which was more highly expressed by
CD4- cells (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cells
Given that cells are commonly involved in host responses to
pathogens (32, 33, 34, 35, 36, 37, 38, 39, 40), it was important to establish the degree to which
cells conform to the Th1/Th2 paradigm. In this regard, Wen et
al. (26) first showed that cells could functionally help B cells
in vivo by production of IL-4, whereafter Ferrick et al. (40) showed
the production of IFN- and IL-4, respectively, by peritoneal and
spleen T cells in response to Th1- and Th2-stimulating
pathogens, respectively. Nonetheless, there was, to date, no
demonstration that these results reflected the differentiated phenotype
of distinct + clones, nor was there a
demonstration of the degree to which various Th1 and Th2 phenotypic
markers cosegregate in clones. These issues are clarified by the
findings presented here that cosegregation of Th1 and Th2 markers
clearly occurs in at least some clones, possibly to a greater
degree than is the case in ß T cell clones (61). This may reflect
the fact that by comparison to ß T cells a greater proportion of
peripheral cells may be preactivated, and hence no longer in a
plastic differentiation state. This would be consistent with our
capacity to readily measure Th1/Th2 cytokine expression by cells
without the need for prior activation (e.g., Fig. 6).
Development of Th1/Th2 cell clones
The generation and maintenance of ß T cell clones is
exclusively dependent on Ag and APCs. Once established, the growth of
the clones reported in this study did not require the sustained
presence of APCs (see Materials and Methods). This
has now been observed in several independent instances. The specificity
of the clones described here is under investigation, but we have
ruled out a requirement for conventional, professional APCs. This is
consistent with >50 characterizations of human and murine TCR
specificity that collectively failed to demonstrate any response to
conventional class I/II MHC-processed peptide (reviewed in 62 .
Hence, the data most strongly suggest that a Th1/Th2 classification can
be established in primary T cells in the absence of specific peptide
presentation by conventional class I/II MHC.
Role for CD4 in the development of Th2 clones
The role of CD4 in the differentiation of Th1 and Th2 ß cells
in CD4-expressing mice has been difficult to assess because CD4 has an
important role during ß T cell development that is epistatic to
the differentiation of peripheral Th1/Th2 cells. This is not the case
for cells, which mostly develop as DN cells. Indeed, recent data
from our laboratory indicates that the rare, but reproducible, numbers
of CD4+ cells that are present in the periphery
of mice (60) and humans (4) develop from CD4-
CD8- thymocytes, not from the
CD4+CD8+ (double positive) pool (63). Two sets
of data in this report suggest that CD4 expression may be more involved
in the differentiation of Th2 cells rather than Th1 cells. First,
although CD4+ cells are rare in vivo (48, 60), the
Th2 clones (albeit only two sibling clones) were both CD4+,
while the Th1 clones were DN. Second, Th2 cytokine RNA expression was
enriched in polyclonal CD4+ cells examined directly
ex vivo, whereas IFN- expression was not. The Ig isotype profiles of
ß T cell-deficient mice also implicate CD4 in Th2 responses; in
TCR-/- mice, some of the B cell help is provided by
CD4+, TCRß+ cells (60, 64). Such T cells
are easily detected in the GCs of this strain (64), and the prevalent
Ig isotypes (e.g., IgG1) are primarily Th2 associated (26, 31). By
contrast, in TCRß-/- mice, all help is provided by
cells (24, 28), most of which are DN. DN CD3+ cells
are readily discernible in the GCs of such mice (28), and the prevalent
Ig isotypes (e.g., IgG2a) are of the Th1 type (24, 28). In summary, the
association of CD4 with Th2 differentiation can be taken to suggest
that systemic cells are more likely to be Th1-type cells than
Th2 cells, consistent with which, IFN- production is more commonly
noted as a product of cells. Nonetheless, there may be an
important biologic role for Th2 cells (see below).
The involvement of CD4 in Th2 differentiation/function may reflect
engagement of an APC by CD4 as well as by TCR , inducing higher
levels of signaling in the responding T cell that are thought to favor
Th2 differentiation (46). This would be consistent with several
observations that CD4 expression is nonetheless not essential for
ß T cells to display a Th2 phenotype (65, 66, 67). Additionally, gut
CD8+ cells have been reported to show Th2-like
activity. In all these instances, Th2 signaling may be induced by high
dose Ag alone and/or by engagement of other molecules in addition to
the TCR.
The only known ligand on APCs for CD4 is MHC class II. Resolving the
specificities of the clones described here will clarify whether
CD4 engagement of MHC class II can augment signaling from a TCR
that is reactive to an MHC class II-independent ligand, or whether the
augmentation only occurs when CD4 and TCR coengage MHC class II,
the latter most likely through a nonconventional mechanism,
previously reviewed (2, 62).
We also note that all the clones tested expressed surface CD1. The role
of CD1 in the immune system is not fully clarified, but there are data
that the direction (Th1/Th2) of an ß T cell response is in part
influenced by the production of cytokines by CD1-reactive NK-T cells.
The data provided here raise the intriguing possibility that
cells might themselves interact with T cells reactive to CD1.
Consistent with this, CD1 was expressed on a subset(s) of cells
in vivo (data not shown), an issue currently under study.
Effector and regulatory functions of + Th
cells in neonates
Collectively, numerous reports have indicated that the number of
cells can greatly increase in humans and/or mice infected with
bacteria, parasites, or viruses, and thus may contribute to the immune
responses to these challenges (32, 33, 34, 35, 36, 37, 38, 39, 40). This may be particularly true
during the neonatal period, when cells are relatively abundant,
and ß T cell-APC interactions may not be fully established. The
clones described here may thus be representative of Th1/Th2
effector cells (40, 41). Indeed, the recent analysis of CD1-/- mice
(68) indicates that significant levels of IL-4 are produced in the
absence of CD1-reactive, NK1.1+ ß T cells,
previously considered as the T cells that skewed ß T cell
responses toward Th2. Given our original findings with cells
(26), Th2 cells might under some circumstances be an important
initiator of Th responses to infection. An important role for Th1/Th2
cells in the establishment of Th cell responses would be
consistent with the impairment in IgA synthesis seen in
TCR-/- mice (69).
At the same time, an increasingly noted phenotype of
TCR-/- mice is one of dysregulated, hyperactive immune
function toward either foreign or self Ags (15, 24, 42, 44). Thus, it
has been inferred from these and other data (43, 45) that cells
ordinarily down-regulate ß T cells of either Th1 (15, 24, 42, 44, 45) or Th2 (43) function, either directly and/or indirectly. Indeed,
T cells have been shown to regulate the activation of
macrophages (70), NK cells (71), and ß T cells (72). It is quite
conceivable that such regulation is mediated by Th1 and Th2 cytokines.
Since exposure to IL-4 of professional APCs, such as macrophages,
reduces their capacity to stimulate Th1 ß cells (reviewed in 21 , it is possible that a major physiologic function of Th2
cells is to attenuate the responses of Th1 ß cells. In this regard
it is notable that although they can be rare, CD4+
cells appear to be conserved in all vertebrates in which they have been
sought. A converse regulatory role (acting on ß Th2 responses) may
prove true for Th1 cells. This hypothesis, that Th1 and Th2
clones play important regulatory roles, would be entirely
consistent with the nonredundant function of cells and ß
cells that is evident from several independent, recently reported
analyses of TCR-/- mice (15, 24, 44).
Note.
During the preparation of this manuscript, two analyses primarily of
the CD4- mouse, indicated that CD4 is also a critical
molecule in Th2 differentiation of ß T cells (76, 77). Such
studies would appear complementary to these studies of cells in
CD4-sufficient hosts.
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Acknowledgments |
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Footnotes |
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2 Present address: Section of Endocrinology, Department of Medicine, Yale University School of Medicine, New Haven, CT 06510.
3 These authors contributed equally to this study.
4 Address correspondence and reprint requests to Dr. Adrian Hayday, Department of Biology, Yale University, KBT 616, 219 Prospect Street, P.O. Box 208103, New Haven, CT 06511.
5 Abbreviations used in this paper: PE, phycoerythrin; FasL, Fas ligand; HPRT, hypoxanthine phoshoribosyl transferase; GC, germinal center; DN, double negative; ICAM-1, intercellular adhesion molecule-1; LPAM, lymphocyte Peyers patch adhesion molecule-1.
Received for publication July 28, 1997. Accepted for publication October 31, 1997.
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