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,
*
Institut de Genetique et de Biologie Moleculaire et Cellulaire, Illkirch, C.U. de Strasbourg, France;
Department of Microbiology and Immunology and
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, TX 77555
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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- and
ß-chains. We have introduced a double mutation that disrupts
interaction with CD4 into the I-Aßk gene and
used this construct to generate transgenic mice expressing only mutant
class II MHC. Although CD4+ thymocytes matured to the
single-positive stage in these mice, their frequency was reduced by
threefold compared with that of wild-type transgenics. Positive
selection of CD4+ T cells in the mutant transgenic mice may
have been mediated by TCRs with a higher than usual affinity for class
II MHC/Ag complexes. In Aßk mutant
transgenics, peripheral CD4+ lymphocytes promoted B cell
differentiation to plasma cells. These CD4+ T cells also
secreted IFN-
in response to various stimuli (e.g., protein Ag,
bacterial superantigen, and alloantigen), but were deficient in IL-2
secretion. Interactions between CD4 and class II MHC molecules appeared
to regulate lymphokine production, with a strong bias toward IFN-
and against IL-2 in the absence of these interactions. Our results have
implications for the manipulation of T cell-dependent immune
responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 complex
(14, 15, 16).
In mice transgenic for a mutant MHC class I molecule that cannot
interact with CD8, it has been unequivocally shown that interactions
between CD8 and MHC class I molecules are required for both positive
and negative selection in the thymus (11, 12, 13). However, the specific
contributions of interactions between CD4 and MHC class II molecules to
thymic T cell selection have not been clearly established. For example,
CD8- T cells can mature to Th1-type, MHC class
II-restricted effector cells in CD4-deficient mice (17). Furthermore,
although there is evidence that CD4-MHC class II interactions
participate in antigenic stimulation (7, 18), Th subset differentiation
(19, 20), and activation-induced cell death (21), it is not known
whether these interactions are indispensable or simply enhance or
accelerate effector functions, perhaps by increasing signals that may
nonetheless be induced in their absence. These questions have been
addressed in MHC class II-deficient mice generated by targeted
disruption of the Aß gene locus in embryonic stem cells
of the H-2b haplotype (22, 23). In mice of this haplotype,
the E gene locus has a natural deletion that results in
the loss of E expression. Therefore, disruption of the
Aß gene eliminates all MHC class II expression in
H-2b mice. Thymic selection of CD4+ T cells is
defective, and almost none of the CD4+CD8+
double-positive (DP) thymocytes mature to CD4+ SP cells in
MHC class II0/0 mice. Although these mice mount efficient
CTL responses against influenza A- or lymphocyte choriomeningitis
virus-infected cells, they do not produce virus-specific Abs (24, 25),
suggesting that Th responses to Ag are absent.
However, CD40/0 and MHC class II0/0 mice do not constitute ideal systems for determining the role of interactions between CD4 and MHC class II in T cell selection and differentiation, because all functions of the deleted molecules are eliminated, rather than just the interaction with one ligand. Thus, an organism that does not express MHC class II molecules is not equivalent to one in which MHC class II molecules are deficient in their ability to interact with CD4 but still able to bind peptides and present to the TCR. Therefore, several groups have attempted to elucidate the role of interactions between CD4 and MHC class II molecules by using mAbs against these cell surface Ags. One caveat of this approach is that anti-CD4 mAbs preferentially deplete CD4+ naive resting T cells (26, 27) and activated memory T cells (27). One mechanism involved in this depletion is apoptosis (28), suggesting that a signal is induced by Ab binding to CD4. Similarly, while mAbs against MHC class II molecules can block interactions with CD4, they also block Ag recognition and interfere with B cell function, inducing signals that can lead to differentiation and apoptosis (29, 30). Thus, Ab-blocking experiments have the disadvantage of inducing disturbances well beyond a simple inhibition of CD4-MHC class II interactions. To circumvent these problems, we decided to generate a transgenic mouse strain that expresses only MHC class II molecules incapable of interacting with CD4.
Interaction with CD4 is mediated by several noncontiguous regions in
both the - and ß-chains of MHC class II molecules (7, 8). One
major site of interaction is located on the ß2 domain of murine MHC
class II molecules (7, 31). Substitution of alanine for glutamic acid
at position 137 of the ß2 domain abolishes the ability of the mutant
MHC class II to induce CD4-dependent T cell functions (7, 8). Because
this site is important for the interaction of MHC class II with CD4, we
chose to mutate amino acid residues glutamic acid 137 and valine 142 in
Aßk, which efficiently forms serologically
detectable, functional heterodimers with Ab
(32). Mice transgenic for the mutant Aßk were
crossed to Aß0/0 mice in the H-2b
background. The only MHC class II molecule expressed in this mouse
strain is an AbAßk
heterodimer. Here, we report characterization of the T cell compartment
and T cell-dependent responses to stimulation with Ag in these mutant
MHC class II-transgenic mice. We demonstrate that despite the inability
of the mutant MHC class II molecules to fully interact with CD4,
CD4+ thymocytes mature and
CD4+CD8- T lymphocytes exist in the periphery,
albeit at a much reduced level compared with that in mice expressing a
wild-type Aßk transgene. Furthermore, the
Aßk mutant transgenic mice can mount
Th-dependent immune responses, and their CD4+ T cells
secrete IFN- but are deficient in IL-2 production compared with
those of wild-type Aßk transgenics.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All chemicals, of the highest purity available, were purchased from Sigma (St. Louis, MO) unless otherwise stated. Staphylococcal enterotoxin A (SEA) was obtained from Toxin Technologies (Madison, WI). Tissue culture media and supplements were purchased from Life Technologies (Gaithersburg, MD).
Generation of transgenic mice
The double mutation Ala for Glu137 and Ala for
Val142 was introduced into Aßk
cDNA using PCR (7). The correct sequence was confirmed by
dideoxynucleotide sequencing from double-stranded templates. The
Aßk wild-type and mutant cDNAs were cloned
into the pDOI plasmid within the rabbit ß-globin gene under the
control of the E promoter (33). Fragments containing the
E promoter, the Aßk wild-type
or mutant cDNA, and a polyadenylation site were excised with
BglI. Fragments free of vector sequences were used for
microinjection into fertilized eggs from (C57BL/6 x
SJL)F2 mice. Founders that transmitted the transgene were
identified by Southern blotting of DNA extracted from tail tissue using
standard techniques (34), and lines were established. Transgenic mice
were bred to Aß0/0 mice. Cell surface
expression was assessed by immunohistochemistry of spleen, lymph node
(LN), and thymus sections and by flow cytometry. The only MHC class II
molecules expressed on the cell surface in these lines are
AbAßk
heterodimers. Lines that displayed normal tissue distribution of
expression were selected. Henceforth, the wild-type
Aßk transgenic line will be referred to as
W+ Aßk, and the mutant line will
be referred to as M Aßk. Transgenic mice were
maintained in a conventional animal care facility (accredited by the
Association for Assessment and Accreditation of Laboratory Animal Care
International) according to the Public Health Service Policy on Humane
Care and Use of Laboratory Animals.
Cell lines and transfections
Murine DAP.3 L cells were transfected with cDNAs encoding
wild-type or mutant E137A.V142A Aßd,
wild-type Ad, and the selective marker
pMo-neo (7). Transfectants were selected as previously described (7),
and then supertransfected with pHßAPr expression vectors (35)
containing cDNAs for ICAM-1 (provided by Dr. Jerry Siu) and B7-1 (gift
from Dr. Ronald Germain) together with the selective marker herpes
simplex virus thymidine kinase, HSVtk/pKS (36). Transfectants were
selected in medium containing G418 (500 µg/ml) and 100 µM
hypoxanthine, 0.4 µM aminopterin, and 16 µM thymidine. Homogeneous
populations were selected by Ab-mediated magnetic bead sorting (7)
using mAbs M5/114 (American Type Culture Collection, Manassas, VA) and
BE29G1 (American Type Culture Collection) against I-Ad and
ICAM-1, respectively. Expression was monitored by flow cytometry on a
FACScan (Becton Dickinson, Mountain View, CA).
Flow cytometric analysis of cell surface Ags
Cell preparations were stained with 40F (mouse anti-Aßk mAb) (37), KT3 (rat anti-mouse CD3 mAb), GK1.5 (rat anti-mouse CD4 mAb), 2.43 (rat anti-mouse CD8 mAb), RA36B2 (rat anti-mouse B220 mAb) (38), H1.2F3 (hamster anti-mouse CD69 mAb; PharMingen, San Diego, CA), or M1/69 (rat anti-mouse HSA mAb; PharMingen). Thymocytes, LN cells, and splenocytes were prepared as described previously (19, 21, 22). Expression of cell surface Ags was determined by one- or two-color flow cytometry on a FACScan (Becton Dickinson). Acquired data were analyzed with the CellQuest program (Becton Dickinson). For three- and four-color analyses, a Coulter ELITE cytometer (Coulter Cytometry, Hialeah, FL) equipped with four-decade logarithmic amplifiers was used.
Ab responses to immunization with protein Ag
All experimental groups consisted of three or four mice. Samples from individual mice were processed separately. All experiments were repeated at least twice. Mice were immunized with 75 µg of keyhole limpet hemocyanin (KLH) emulsified in CFA and were bled 7 and 10 days later. Twenty days after immunization, mice were boosted by i.p. injection with 25 µg KLH in PBS, and were again bled seven days later. KLH-specific Abs were quantified by ELISA as previously described (19), using Abs specific for mouse IgM, IgG, IgG1, and IgG2a.
T cell functional assays
Cellular proliferation of LN T cells (LNTC). Axillary, brachial, cervical, inguinal, and popliteal LNs were dissected from KLH-immunized mice. Cell suspensions were prepared and depleted of MHC class II+ and CD8+ cells with mAb 39E and 2.43, respectively (19). LNTC (2 x 105) were incubated with 4 x 105 irradiated (3000 rad) splenocytes from either W+ Aßk or M Aßk nonimmunized transgenic mice and various concentrations of KLH in the wells of a flat-bottom 96-well plate. Culture supernatants were harvested after 24, 48, or 72 h for the determination of cytokine contents. After 72 h, [3H]thymidine was added for an additional 16 h of incubation. Thereafter, the cells were harvested on a 96-well cell harvester, and incorporated radioactivity was measured in a Packard Direct Matrix beta counter (Packard, Downers Grove, IL).
Cellular proliferation of splenocytes. Splenocytes (4 x 105) from KLH-immunized mice were incubated in the wells of a flat-bottom 96-well plate with various concentrations of KLH. Supernatants and cells were processed as described for LNTC.
Cytokine production.
IFN- titers were measured by ELISA in 48-h culture supernatants
(19). IL-2 secretion was measured by determining the ability of a 1/4
dilution of 24-h culture supernatants to support the growth of the
IL-2-dependent cell line CTLL-2 (19). To establish optimal timing for
measuring cytokine secretion, culture supernatants harvested 24, 48, or
72 h after stimulation were tested for both IFN- and IL-2.
In vitro stimulation with SEA. Axillary, brachial, cervical, inguinal, popliteal, and mesenteric LNs were dissected from unprimed mice. LN cell preparations were depleted with mAb 39E. LNTC (4 x 105) were incubated with 5 x 104 L cell transfectants expressing either wild-type or mutant I-Ad together with ICAM-1 and B7-1. Various concentrations of SEA were added to the cultures. Cytokine production and cellular proliferation were measured as described above for KLH-restimulated LNTC.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although cell surface expression was low, W+
Aßk and M Aßk lines
were well matched. In heterozygote transgenic mice, the level of
W+ Aßk and M
Aßk expression on peripheral B cells was only
about 5% that in B10.A control mice (homozygous for
AkAßk). Breeding
mice homozygous for the transgenes increased cell surface expression of
W+ Aßk and M
Aßk to about 10% of that found in B10.A
controls (Fig. 1
). Expression of
W+ Aßk and M
Aßk in the thymus paralleled that found on
peripheral B cells. Importantly, all B220+ B cells also
expressed the transgene. In both W+
Aßk and M Aßk mice,
we found normal tissue distribution of transgene expression (data not
shown).
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To determine whether positive selection of SP CD4+
thymocytes occurred in M Aßk mice, we
measured the percentages of double-negative, DP, and SP thymocytes.
Fewer SP CD4+ cells were present in the thymi of M
Aßk mice than in those of W+
Aßk mice. The population of CD4+
thymocytes was, however, slightly larger in M
Aßk than in Aß0/0
mice (Table I). To more carefully analyze
T cell maturation, we assessed CD69 and HSA expression. CD69 and HSA
are markers for maturing and mature thymocytes, respectively. Thymi of
M Aßk mice contained considerably more
maturing (Fig. 2) and mature (Fig. 3) CD4+ T cells than did
those of Aß0/0 mice, but fewer than those of
W+ Aßk mice. This was confirmed
by treating mice with dexamethasone (Table II), a substance that induces apoptosis
in immature thymocytes. That more mature CD4+ T cells were
present in W+ Aßk than in M
Aßk thymi indicates that the mutation had a
strong effect on the selection of CD4+ T cells. It is,
however, evident that a small number of CD4+ cells fully
matured in M Aßk thymi.
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ß TCRs
have been reported in MHC class II-deficient mice (22) and are a
consequence of deficient interactions between CD4 and MHC class II
molecules (39, 40). Peripheral T lymphocytes
To confirm that CD4+ T cells fully matured in M
Aßk mice, we measured CD4 and CD8 expression
on LNTC. Importantly, LN from M Aßk mice
contained a significantly larger proportion of CD4+ cells
than did LN from Aß0/0 mice (Table I
).
Furthermore, CD3 expression was higher on M
Aßk than on Aß0/0
CD4+ LNTC (data not shown). Analysis of the TCR repertoire
in peripheral T cells from M Aßk mice showed
a normal distribution. In particular, the increase in Vb5 and the
decrease in Vb4- and Va2-expressing cells that is characteristic of
Aß0/0 mice (41) did not occur in M
Aßk mice (data not shown).
Primary and secondary Ab responses to KLH
After we had established that CD4+ thymocytes fully matured and were positively selected in M Aßk mice, we initiated experiments to determine whether CD4+ T lymphocytes from M Aßk mice could respond to Ag. Mice were immunized with 75 µg of KLH emulsified in CFA and bled 7–10 days later. Twenty days after immunization, mice were boosted by i.p. injection with 25 µg of KLH in PBS and were again bled 7 days later. KLH-specific Abs were quantified by ELISA as previously described (19), using Abs specific for mouse IgM, IgG, IgG1, and IgG2a.
Amounts of primary and secondary anti-KLH Abs (IgM plus IgG) were
similar in W+ Aßk mice and
littermate controls, whereas primary Ab responses in M
Aßk mice were much lower (Fig. 5). However, M
Aßk, but not Aß0/0,
mice produced secondary anti-KLH Ab titers comparable to those in
W+ Aßk mice. We then measured the
progression of anti-KLH IgM and IgG titers in W+
Aßk and M Aßk mice
homozygous for the transgene. Titers of anti-KLH IgG were 5- to
10-fold lower in M Aßk than in W+
Aßk mice 7 and 10 days after primary
immunization (Table III). Whereas 7 days
after priming, IgM titers also differed by 5- to 10-fold between
the two lines, only a 2-fold difference was detected on day 10 after
immunization. Therefore, M Aßk mice were
capable of generating both anti-KLH IgM and IgG after priming,
but with slower kinetics than did W+
Aßk mice. After a booster immunization, IgM
and IgG titers against KLH did not differ significantly between
W+ Aßk and M
Aßk mice. Also, the amounts of IgG1 did not
differ between W+ Aßk and M
Aßk transgenic mice (Table III). Thus,
CD4+ T lymphocytes selected and primed in M
Aßk transgenic mice could provide help for B
lymphocytes and promote Ig class switching.
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-induced expression of the IgG2a isotype is often used to
characterize Th1-type responses, whereas IL-4-dependent expression of
the IgG1 isotype characterizes Th2-type responses. However, in mouse
strains with the Igh1-b allele, such as the background
strains C57BL/6 and SJL used in our experiments, the gene for IgG2a is
deleted (42). Therefore, we could not use measurements of IgG2a as an
indicator of Th1 responses. In vitro recall response after primary immunization
LN and splenic T cells from KLH-primed W+
Aßk and M Aßk mice
responded to in vitro stimulation with the Ag. At low Ag
concentrations, dependence of KLH-specific T lymphocytes on effective
interactions between CD4 and MHC class II was apparent, because
CD4+ LNTC from both W+
Aßk and M Aßk mice
responded better to Ag presented by W+
Aßk than by M Aßk
APCs (Fig. 6A). Splenocytes from either W+
Aßk or M Aßk mice
proliferated equally well upon restimulation in vitro (Fig. 6B).
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upon in vitro restimulation with KLH presented by either
W+ Aßk or M
Aßk APC (Fig. 7). APC from W+
Aßk mice induced significantly higher IFN-
secretion in LNTC from both W+
Aßk and M Aßk mice
than did M Aßk APC. It is therefore likely
that CD4 molecules on LNTC from M Aßk mice
were able to engage wild-type
AbAßk upon
antigenic stimulation and function appropriately.
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After a second immunization, splenocytes from W+ Aßk transgenic mice responded to in vitro restimulation with KLH at 500 µg/ml with a stimulation index of 10.18 ± 4.02 (n = 4), whereas the stimulation index for splenocytes from M Aßk transgenic mice was 4.66 ± 1.18 (n = 3; p = 0.074). No significant differences were observed in the proliferation rate of LNTC from KLH-primed and boosted W+ Aßk and M Aßk transgenics. As observed after primary immunization, at low Ag concentrations LNTC from both transgenic strains responded better to Ag presented by W+ APC.
Following boosting, LNTC from M Aßk mice
secreted more IFN- than did LNTC from W+
Aßk mice after stimulation with KLH presented
by either W+ Aßk or M
Aßk APC (Fig. 7). On the other hand,
detectable levels of IL-2 were measured from supernatants of LNTC from
W+ Aßk mice stimulated with 5 or
50 µg/ml KLH presented by W+
Aßk or M Aßk APC,
but IL-2 secretion by LNTC from M Aßk mice
was barely above the background level (data not shown). Similarly,
splenocytes from W+ Aßk mice
secreted significant amounts of IL-2 upon restimulation in vitro with
various doses of KLH, whereas splenocytes from M
Aßk mice did not (Fig. 8). IL-4 titers were
generally very low in either strain (data not shown). For the KLH
restimulation experiments, LN cells were depleted of class II
MHC+ and CD8+ cells, and therefore, equivalent
numbers of CD4+ LNTC from W+ and M
Aßk transgenic mice were used. Splenocytes
were not depleted, and the percentages of CD4+ T cells in
splenic preparations from W+ Aßk
and M Aßk mice were 13.4 ± 1.1
(mean ± SD; n = 6) and 3.5 ± 1.4%,
respectively. However, differences in IL-2 secretion cannot be
explained by differences in the percentage of CD4+ T cells,
because splenic preparations from M Aßk mice
never secreted any detectable IL-2 (measured in supernatants harvested
24, 48, or 72 h after restimulation).
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Because IL-2 secretion by LNTC from M
Aßk mice in response to KLH restimulation was
reduced compared with that by LNTC from W+
Aßk mice, but IFN- secretion was
relatively enhanced, it is unlikely that different frequencies in
KLH-responsive T cells caused the dichotomy in cytokine production.
Thus, either properties intrinsic to the LNTC or differences in priming
by W+ Aßk or M
Aßk APC induced divergent antigenic
responses. Therefore, we measured the ability of naive LNTC derived
from the two strains of mice to respond to superantigen presented by
wild-type or mutant MHC class II (Fig. 9). LN cells were depleted of
MHC class II-expressing cells and incubated with APC and SEA. To
eliminate any possible contamination of the cultures with splenic T
cells when replenishing with APC, we used L cell transfectants
expressing either wild-type or mutant I-Ad as APC. APC
expressing mutant I-Ad elicited equivalent proliferative
responses in LNTC from W+ Aßk and
M Aßk mice. LNTC from W+
Aßk mice responded more vigorously to SEA
presented by APC expressing wild-type I-Ad than did LNTC
from M Aßk mice. However, the difference in
IL-2 secretion between LNTC from W+
Aßk and M Aßk mice
was much more pronounced. Whereas LNTC from W+
Aßk mice secreted IL-2 in response to the
allogeneic stimulation by I-Ad, LNTC from M
Aßk mice did not respond to the allogeneic
stimulus. Furthermore, LNTC from W+
Aßk mice responded to as little as 5 ng/ml of
SEA with increased IL-2 secretion, but LNTC from M
Aßk mice required at least 2 µg/ml of SEA
to secrete any detectable IL-2. Thus, LNTC from M
Aßk mice appear to be deficient in the
ability to secrete IL-2 upon stimulation with SEA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 3, and Table II) and peripheral CD4+ lymphocytes (Fig. 4). However, whereas positive selection
of CD4+ thymocytes is almost obliterated in
Aß0/0 mice, it was only reduced by about
threefold in M Aßk compared with
W+ Aßk transgenics (Tables I and
II). The peripheral CD4+ T lymphocytes in M
Aßk mice were functional with regard to their
ability to provide B cell help for the generation of KLH-specific Abs,
although the pace of their response was delayed (Fig. 5 and Table III). Furthermore,
CD4+ T lymphocytes from M Aßk
mice proliferated and produced IFN- in response to immunization and
restimulation with KLH (Figs. 6 and
7).
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Our system excluded the possibility of CD4 and TCRs interacting with
different MHC class II molecules, because the only MHC class II
expressed was the endogenous Ab paired with
the transgenic wild-type or mutant Aßk.
Further, because we analyzed a polyclonal T cell population, effects of
the mutation in Aßk on binding to CD4 could
not be masked by high affinity binding of a single TCR to the mutant
MHC class II. Rather, our results suggest that some, but not all,
TCR-MHC class II/Ag interactions depend on participation of CD4 in the
trimeric complex. Therefore, it is possible that at least some of the
positively selected CD4+ T cells may express TCRs that
would cause deletion in a thymus with wild-type MHC class II. However,
whereas CD4+ LNTC from M Aßk mice
proliferated in response to BALB/c APC (not shown), they failed to
mount an MLR against W+ Aßk APC
(see Fig. 6). This is in contrast to observations made in mice
transgenic for a mutant MHC class I molecule that is incapable of
interacting with CD8. These mice are deficient in negative selection
and consequently do not delete CD8-dependent T cells reactive against
the wild-type MHC class I (11, 12, 13). Therefore, negative selection of
maturing CD4+ T cells may not be stringently controlled by
interactions between CD4 and MHC class II molecules.
Riberdy et al. (40) recently reported on the development of CD4+ T cells in mice expressing a mutant Aßb transgene. Their results are similar to our findings, in that drastically reduced numbers of conventional CD4+ T cells are observed in this system. In addition, thymocytes expressing the AND transgenic TCR are not positively selected in mutant Aßb transgenic mice (40). In another system described by Fuller-Espie et al. (43), chimeric MHC class II transgenes expressing HLA-DR ß1 and I-E ß2 domains on a Aß0/0 background also positively selected CD4+ thymocytes. These investigators suggested that positive selection may be sensitive to quantitative variation in MHC class II density, unmasked when Ag is limiting, but relatively insensitive to qualitative variation in the MHC class II-CD4 interaction. It is, however, possible that DR ß1/E ß2 chimeric molecules efficiently interact with CD4, and that MHC class II-CD4 interactions may not be perturbed in this system.
We also tested Ag-specific T cell responses in mutant and wild-type MHC
class II transgenic mice. In our experiments, peripheral
CD4+ T lymphocytes from M Aßk
transgenics were able to promote isotype switching in response to T
cell-dependent B cell Ags (Fig. 5 and Table III). However, the ability
to mount primary Ab responses was severely curtailed in M
Aßk transgenics, whereas W+
Aßk mice did not differ from normal controls.
We also observed a drastically decreased ability of T cells from
KLH-primed M Aßk mice to produce IL-2 upon in
vitro restimulation with Ag (Fig. 8).
This defect was not reflected in a concomitant decrease in
proliferation following antigenic stimulation (Fig. 6). Similarly,
naive M Aßk LNTC responded less well than
W+ Aßk to stimulation with the
bacterial enterotoxin, SEA. Again, IL-2 secretion was drastically
affected, and proliferation only moderately so (Fig. 9). The observed defect in IL-2 secretion
upon activation was not due to a more general defect in responsiveness,
because in vitro restimulation elicited similar IFN- responses in
KLH-primed LNTC from either strain of mice. Further, after an
additional booster injection with KLH, CD4+ M
Aßk LNTC secreted considerably more IFN-
than did CD4+ W+ LNTC (Fig. 7).
Our findings suggest that Ag-primed T cells in M
Aßk transgenic mice predominantly
differentiate to IFN--secreting effectors. Other experimental
systems lead to similar conclusions: 1) the development of Th lineage
cells to Th2 effector cells is compromised in CD40/0 mice,
but maturation to Th1 cells occurs (17); 2) priming of CD4+
T cells by APC that express mutant MHC class II unable to interact with
CD4 promotes differentiation to the Th1 type and prevents the
development of cells secreting IL-4 (20); and 3) peptides that mimic
the CD4 binding site on MHC class II and interfere with CD4-MHC class
II interactions promote Th1 effector cells in KLH-primed BALB/c mice
(19). Therefore, CD4-MHC class II interactions may differentially
regulate T cell signaling pathways. Whereas IL-2 gene up-regulation
depends on signals induced by CD4-MHC class II interactions (7, 44),
IFN- secretion either is independent of these interactions or may
even be inhibited. Because CD4-MHC class II interactions during T cell
activation may promote differentiation to Th2 effectors, inhibition of
IFN- secretion could be an indirect effect mediated via increased
IL-10 synthesis by Th2 cells (45).
In conclusion, we have generated a transgenic mouse model for determining the role of interactions between CD4 and MHC class II molecules in vivo. All mature CD4+ T cells present in these mice were selected on MHC class II molecules that are incapable of interacting with CD4. Our results on the function of CD4+ T lymphocytes in these mutant MHC class II transgenic mice further suggest that affecting CD4-MHC class II interactions during antigenic stimulation may be a means to manipulate Th cell responses.
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Acknowledgments |
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Footnotes |
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2 Current address: Basel Institute for Immunology, Grenzacherstrasse 487, 4005 Basel, Switzerland.
3 Address correspondence and reprint requests to Dr. Rolf König, Department of Microbiology and Immunology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1070.
4 Abbreviations used in this paper: SP, single positive; DP, double positive; SEA, staphylococcal enterotoxin A; KLH, keyhole limpet hemocyanin; LN, lymph node; LNTC, lymph node T cell.
Received for publication May 14, 1998. Accepted for publication August 28, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß chains in CD4 function indicates a role for ordered oligomerization in T cell activation. J. Exp. Med. 182:779.ß T-cell receptor determine the CD4/CD8 phenotype of T cells. Nature 335:229.[Medline]
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3 domain: CD8 and the T-cell receptor recognize the same class I molecule. Proc. Natl. Acad. Sci. USA 87:2137.This article has been cited by other articles:
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  H.-J. Kim, D. Guo, and D. B. Sant'Angelo Coevolution of TCR-MHC interactions: Conserved MHC tertiary structure is not sufficient for interactions with the TCR PNAS, May 17, 2005; 102(20): 7263 - 7267. [Abstract] [Full Text] [PDF]
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 H. Kao and P. M. Allen An antagonist peptide mediates positive selection and CD4 lineage commitment of MHC class II-restricted T cells in the absence of CD4 J. Exp. Med., January 3, 2005; 201(1): 149 - 158. [Abstract] [Full Text] [PDF]
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 T. L. Denning, H. Qi, R. Konig, K. G. Scott, M. Naganuma, and P. B. Ernst CD4+ Th Cells Resembling Regulatory T Cells That Inhibit Chronic Colitis Differentiate in the Absence of Interactions Between CD4 and Class II MHC J. Immunol., September 1, 2003; 171(5): 2279 - 2286. [Abstract] [Full Text] [PDF]
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Q. Wang, L. Malherbe, D. Zhang, K. Zingler, N. Glaichenhaus, and N. Killeen CD4 Promotes Breadth in the TCR Repertoire J. Immunol., October 15, 2001; 167(8): 4311 - 4320. [Abstract] [Full Text] [PDF]
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P. A. Sieling, M.-T. Ochoa, D. Jullien, D. S. Leslie, S. Sabet, J.-P. Rosat, A. E. Burdick, T. H. Rea, M. B. Brenner, S. A. Porcelli, et al. Evidence for Human CD4+ T Cells in the CD1-Restricted Repertoire: Derivation of Mycobacteria-Reactive T Cells from Leprosy Lesions J. Immunol., May 1, 2000; 164(9): 4790 - 4796. [Abstract] [Full Text] [PDF]
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R. Maroto, X. Shen, and R. Konig Requirement for Efficient Interactions Between CD4 and MHC Class II Molecules for Survival of Resting CD4+ T Lymphocytes In Vivo and for Activation-Induced Cell Death J. Immunol., May 15, 1999; 162(10): 5973 - 5980. [Abstract] [Full Text] [PDF]
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