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Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice expressing single AbEp complex with invariant chain (AbEpIi+)3 or without invariant chain (AbEpIi-) and deficient in the expression of wild-type Ab molecules were generated at the National Jewish Medical and Research Center (Denver, CO). These animals and MHC class II-deficient (Ab-), AbEpIi-ß2m-, and F1 (AbEpIi- x Abwt) mice were further bred in the animal facility at the Medical College of Georgia (Augusta, GA). Animals deficient in MHC class I and II (MHC-) and animals deficient in CD4 molecules were also bred in the above facility.
C57BL/6 and congenic mice B6.PL-Thy-1a/Ca expressing the CD90.1/Thy-1.1 Ag were purchased from The Jackson Laboratory (Bar Harbor, ME).
Radiation bone marrow chimeras
Bone marrow from donor mice was depleted of mature T cells using anti-Thy-1.2 mAb (HO 13.4.6) and complement and was subsequently checked for purity by FACS. Recipient mice were lethally irradiated (1100 R) and reconstituted within 6 h with 5 x 106 cells. If not otherwise stated, radiation chimeras were used for experiments at least 3 wk postreconstitution.
Complement depletion
Single cell suspensions from pooled axillary, inguinal, mesenteric, and paraaortic lymph nodes were prepared and incubated for 30 min at 4°C with cytotoxic mixture prepared from supernatants or with purified Abs from the following hybridomas cultured in this laboratory: anti-CD8 (clone HO 2.2), anti-MHC class II (clones 25-6-3S and BP107.2.2), anti-CD45 (clone B220), and antiJ11D (clone J11D.2). Cells were then incubated at 37°C with a mixture of rabbit (Sigma, St. Louis, MO) and guinea pig (Life Technologies, Grand Island, NY) complement. After a single washing, cells were checked by flow cytometry for the presence of cells bearing CD8 or class II MHC (Ab). Purity of more than 90% CD4+ cells was achieved with no Ab cells present.
In vitro stimulation
Plastic flasks were coated overnight with anti-TCR Ab (HAM 57.597.2) in balanced salt solution and washed once. Purified CD4+ T cells were cultured in them for 6 days with addition of IL-2 on day 3.
Cell tracking
For tracking lymphocytes in vivo, a lipophilic succinimidyl ester of carboxyfluorescein diacetate (CFSE) purchased from Molecular Probes (Eugene, OR) was used. CFSE passively enters the cytoplasm, where it is rapidly hydrolyzed into a fluorescent hydrophilic metabolite that is unable to diffuse out and is readily detectable by FACS. CFSE concentration decreases with each cell division.
Adoptive transfers
CD4+ lymphocytes, purified by complement depletion, were suspended at 5 x 106/ml in PBS, and CFSE was added to a final concentration of 1 µg/ml and incubated at 37°C for 15 min. Cells were washed, suspended in PBS, counted, and injected i.v. into recipient animals, which, if not otherwise stated, had been sublethally (600 rad) irradiated.
Flow cytometry
The biotin-, fluorescein-, or PE-labeled mAbs were prepared in this laboratory or purchased from PharMingen (San Diego, CA). Cells were suspended in blocking solution (50% culture supernatant of anti-FcR Ab 2.4G2 and 50% FBS) and incubated (4°C, 30 min) with Abs of interest washed twice and analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Diego, CA).
Quantitation of total cell number
Single cell suspensions were prepared from the pooled axillary, inguinal, mesenteric, and paraaortic lymph nodes and spleen (treated with ammonium chloride buffer to lyse erythrocytes), and cell numbers were measured. Percentage of CD4+ T lymphocytes of interest (depending on experiment: CFSE+, CFSE-, Thy-1.1+, Thy-1.1-) in each animal was evaluated using flow-cytometric analysis. Total number was obtained by adding the number of cells in spleen and twice the number found in lymph nodes, as described (18, 19). For the purpose of these calculations, all cells outside of the clearly negative group were considered to be CFSE+. There were lymphocytes with high and low levels of fluorescence among them, but no such distinction was made.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study was undertaken to determine whether the
diversity of self-derived peptides bound to class II MHC influences the
persistence of CD4+ T cells in the periphery as
it does in the thymus. It is known that naive
CD4+ T cells continuously sense the presence of
self MHC molecules in the periphery, but whether particular self
peptides bound to MHC are also specifically recognized during these
interactions has not been investigated. To approach this question, we
intended to use, as recipients for adoptively transferred wild-type
CD4+ T cells, mice that lack endogenous
Abß-chain together with an Ii chain and that
are transgenic for Abß covalently attached with
single peptide E
(52–63)
(AbEpIi- mice). A
potential caveat for using
AbEpIi- mice in these
experiments is a lower expression of the transgenic single
AbEp complex in comparison with the
Ab bound with endogenous peptides in wild-type
mice (20). Hence, one may be concerned that the expression
level of AbEp complex may be insufficient for
CD4+ T cells that developed in mice expressing a
normal level of Ab. Therefore, to test whether
lower expression of the transgenic Ab will
support the survival of wild-type CD4+ T cells if
bound with endogenous self peptides, we transferred these T cells into
transgenic AbEpIi+ mice. In
the AbEpIi+ mice, the
covalent AbEp complex is unstable due to an Ii
chain that directs the AbEp to endosomes, where
covalent peptide is cleaved and replaced with the diverse set of
self-derived peptides (21). Because of that,
AbEpIi+ mice that
express the same lower levels of transgenic Ab as
AbEpIi- mice select and
maintain a high number of CD4+ T cells in the
thymus and in the periphery (20). As shown in Fig. 1
, wild-type CD4+ T
cells labeled with fluorescent dye (CFSE) and adoptively transferred to
sublethally irradiated
AbEpIi+ mice or to
wild-type mice proliferated with similar kinetics (10 and 30 days after
transfer), indicating that transferred CD4+ T
cells perceive both low and high numbers of Ab if
bound with many peptides. This result indicated that transgenic
Ab bound to self peptides is sufficient to
support the survival of adoptively transferred
CD4+ T cells from mice expressing
Abwt.
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Next, we sought to determine whether CD4+ T
cells that matured in wild-type mice would survive in the presence of a
single AbEp complex. Three different groups of
sublethally irradiated recipients, differing in the complexity of self
peptides bound to Ab, were used. The first group
of mice lacked expression of CD4 and had no CD4+
T cells in the periphery, but expressed intact class II MHC molecules
(Ab) loaded with a diverse set of wild-type
peptides (22). The second group of mice was
AbEpIi- mice with all
Ab molecules covalently bound with the single
peptide (16). The third type of recipient mice had no
expression of Ab molecules and very few
peripheral CD4+ T cells (23). All
these groups of mice were injected with CFSE-labeled wild-type
CD4+ T cells bearing a different allele of Thy-1
Ag to follow their fate regardless of CFSE label. As shown on the
left panel in Fig. 2A, after 2 days, all
detectable CD4+ Thy-1.1+ T
cells were positive for CFSE in all recipients. Four weeks later,
one-third of CD4+ Thy-1.1+
T cells transferred to Abwt mice were CFSE
positive, while 75% and 88% remained CFSE positive in the
AbEpIi- or
Ab- recipients, respectively. After 30 days,
recipients were sacrificed and the total numbers of recovered
CD4+ Thy1.1+ T cells were
calculated. We found that transferred CD4+ T
cells in wild-type mice significantly expanded, whereas in the two
other types of recipients, the same transferred cells decayed by more
than 70% (Fig. 2B). The number of
CSFE+ T cells was also highest in the
Abwt recipients because this population included
many continuously expanding cells that had not yet completely lost the
CSFE staining (Fig. 2B). As shown in Fig. 3A, 90% of
CD4+ T cells isolated from B6.PL mice had a naive
phenotype
(CD44lowCD45high), but when
transferred into syngenic wild-type recipients, the expanding cells
up-regulated CD44 and down-regulated CD45RB expression (Fig. 3, B and C). In contrast, in two other types of
recipients that expressed a single class II MHC/peptide complex or had
no class II MHC, the majority of transferred CD4+
Thy-1.1+ cells decayed after 30 days with at most
an unchanged phenotype. Expression of activation markers after adoptive
transfer of naive TCR transgenic, CD4+ T cells
into a syngenic irradiated recipient has been previously reported
(24, 25). These cells sustained elevated expression of
CD44 in the absence of Ag for few weeks. Our experiments confirm this
phenomenon, and additionally show that to survive and to expand, naive
wild-type CD4+ T cells require a diverse set of
self-derived peptides bound to class II MHC molecules.
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The fact that the majority of wild-type derived CD4+ T cells quickly disappeared when transferred into the in vivo environment of class II MHC preloaded with one peptide contrasted with the extended survival of naive CD4+ T cells present in the single class II MHC/peptide mice (Ref. 16 ; P. Muranski, unpublished data). This observation could indicate that one of the self Ab/peptide complexes that may provide the survival signal to naive CD4+ T cells is the same self-derived peptide(s) that bound to the class II MHC that positively selected them in the thymus. To test this hypothesis, we have performed two adoptive transfer experiments.
In the first experiment, we isolated CD4+ T cells
from ß2-microglobulin-deficient wild-type mice
(Abwtß2m-)
or mice expressing only AbEp complex
(AbEpIi-ß2m-),
labeled them with CSFE, and separately adoptively transferred them into
sublethally irradiated
AbEpIi-ß2m-
recipients. Two days after transfer, bright
CD4+CSFE+ T cells from both
types of donors were present at significant amounts in recipients
expressing AbEp complex (Fig. 4). In contrast, 2 wk after transfer,
most of CSFE+CD4+ T cells
from the
AbEpIi-ß2m-
donors were proliferating and declining their CSFE staining, while
CD4+ T cells from the
Abwtß2m-
donors divided once or not at all. Four weeks after transfer, multiple
divisions of
AbEpIi-ß2m--derived
CD4+ T cells were even more pronounced. These
results implied that the AbEp complex supports
the expansion of self-selected CD4+ T cells, but
is unable to support survival of CD4+ T cells
that developed in mice expressing Ab molecules
bound with many self peptides.
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, virtually all
CD4+ T cells were CFSE positive 2 days after
transfer, whereas on day 9, the majority of transferred cells were
already CFSE negative, regardless of their origin. However, in less
than 3 wk, the population of transferred
CD4+Thy-1.1- T cells
almost completely lost the CFSE staining due to proliferation, while
the majority of
CD4+Thy-1.1+ T cells
remained CFSE positive. When the total number of transferred
CD4+ T cells was calculated, we found that the
CD4+ T cells derived from
AbEpIi- mice expanded
several times, while CD4+ T cells derived from
wild-type mice were almost completely extinct (Fig. 5B). In
previous experiments, the same wild-type CD4+ T
cells expanded without restraint if transferred into mice expressing
Abwt in the periphery (Figs. 2
and 3), proving
that they can proliferate in the appropriate MHC/peptide environment.
Hence, we concluded that to survive and expand, naive
CD4+ T cells favor contact with the MHC/peptide
complexes that were encountered during their thymic selection.
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The survival of naive and Ag-experienced T cells in the periphery
is regulated differently, and both populations are believed to occupy
nonoverlapping ecological niches (26). Memory
CD8+ T cells transferred into host with
nonrestricting class I MHC survived for the extended period of time
(19), but decayed in 1 wk in
Db-Kb-ß2m--deficient
mice (27), implying that effector/memory T cells may not
tightly depend on the expression of a particular type of MHC.
Similarly, it was reported that in vitro generated effector
CD4+ T cells transferred into mice that do not
express specific Ag survived for several weeks (7, 28).
However, whether activated CD4+ T cells require
particular self peptides bound to MHC to persist, as naive cells do,
has not been investigated. To assess whether self peptides bound to
Ab influence the survival of activated
CD4+ T cells, we transferred in vitro activated
CD4+ Thy-1.1+ T cells into
sublethally irradiated recipients that lack Ab or
express Ab bound with one or with many peptides.
The effector CD4+ Thy-1.1+
T cells were generated from lymph node-derived naive
CD4+ T cells during 6 days of incubation in the
presence of IL-2 and immobilized mAb specific for ßTCR. Activated
CD4+ Thy-1.1+ T cells were
then labeled with CFSE, and adoptively transferred into sublethally
irradiated recipients. As shown in Fig.
6A, 2 days after transfer,
all CD4+ Thy-1.1+ cells
remain CFSE positive in all groups of recipients. However, when
recipients were sacrificed 30 days after transfer, the majority of
CD4+ Thy-1.1+ T cells
recovered from wild-type mice were CFSE negative, in contrast to two
other recipients in which more than one-half of the transferred cells
retained the dye. As shown in Fig. 6B, the number of
recovered CD4+ Thy-1.1+ T
cells increased in wild-type recipients concurrent with the staining
data, implying that to expand, wild-type derived effector
CD4+ T cells may favor expression of multiple
endogenous peptides bound to Ab. The number of
CD4+ Thy-1.1+ T cells
recovered from the two other recipients was close to the number
transferred initially, and one-half of them were CFSE negative,
indicating that at least a portion of transferred effector
CD4+ T cells expanded regardless of the presence
or absence of class II MHC.
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A, 2 days after
transfer, Thy-1.1- and
Thy-1.1+ CD4+ T cells were
all CFSE positive. However, on day 9, 31% of transferred
CD4+ Thy-1.1- cells, vs
83% of Thy-1.1+ T cells, retained the dye.
Similar proportions of CFSE-positive and CFSE-negative cells were found
after 21 days, when recipients were sacrificed and the number of
recovered cells was calculated (Fig. 7B). Activated
CD4+ Thy-1.1- cells from
AbEpIi-ß2m-
mice almost doubled, whereas the wild-type derived effector
CD4+ T cells did not decay, but also did not
expand. Therefore, we conclude that low affinity contact with self
class II MHC/peptide ligand(s) may be required by some of activated
CD4+ T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we show that the majority of naive CD4+ T cells selected in wild-type mice and adoptively transferred into mice expressing a single class II MHC/peptide complex quickly diminished, with similar kinetics as reported for CD4+ T cells seeded into mice that lack class II MHC molecules (12). Hence, this result implies that CD4+ T cells use their TCRs to sense self peptides bound to MHC molecules not only during selection in thymus, but also in the secondary lymphoid organs, where these interactions maintain prolonged survival of the T cells.
What are the features of the self peptides that uphold viability of naive T cells in the periphery and what is their relationship to the self peptides that select the same T cells in the thymus? One answer to this question is that selecting MHC/peptide may be one of the MHC/peptide complexes able to maintain the persistence of these T cells in the periphery. The similarity of the MHC/peptide(s) ligand responsible for the selection and maintenance of the clonotypic TCR has been implicated in several reports, but experimental data proving this assumption have not yet been convincingly presented (24, 25, 33, 34). In our experiments, transfer of naive CD4+ T cells from AbEpIi- or wild-type mice into lethally irradiated Ab- mice reconstituted with bone marrow from AbEpIi- mice resulted in the preferential expansion of only CD4+ T cells from AbEpIi- mice. In contrast, wild-type derived naive CD4+ T cells disappeared at large by 3 wk after transfer, indicating that AbEp complex supports primarily the survival of CD4+ T cells selected by this complex. The kinetics of the decay of wild-type CD4+ T cells was slightly accelerated in the presence of CD4+ T cells derived from AbEpIi- mice, implying that competition between peripheral T cells may also have some influence on their survival. We are currently testing this idea using CD4+ T cells with clonotypic TCRs positively selected by different class II MHC/peptide complexes. Admittedly, a single class II MHC/peptide complex does not match the high expression level of Ab molecules bound with self peptides present in wild-type mice. Therefore, one may argue that if the survival signal for naive CD4+ T cells is calibrated by thymic selection, discrepancy in the expression level of Ab encountered by the same T cell in the thymus or periphery may result in its shortened survival (35) However, naive CD4+ Thy-1.1+ T cells exposed in vivo to low levels of transgenic Ab loaded with endogenous peptides in the presence of Ii did not lead to the extinction of transferred T cells for 4 wk after transfer. Hence, it seems that the low level of transgenic Ab is sufficient to maintain the survival of naive wild-type CD4+ T cells only if bound with a diverse spectrum of self-derived peptides. Limited survival of wild-type CD4+ T cells transferred into the AbEpIi- mice was also not a result of adverse rejection mediated by NK cells, because hosts were compromised by sublethal irradiation before transfer and some of transferred CFSE-positive T cells remained detected in AbEpIi- mice after 5 wk. Experimental parameters such as the dose of irradiation, number of transferred cells, and duration of the experiment are critical in revealing the proliferation of transferred CD4+ T cells in recipients expressing class II MHC bound with low affinity peptide ligand. Hence, the differences in these settings may be responsible for the distinct conclusion obtained in the similar studies (34). In this study, to show that naive CD4+ T cells require low avidity contacts with self MHC/peptide complexes to survive in the periphery, we transferred 3 x 106 of naive CD4+ T cells and followed their gradual expansion for several weeks. This length of time was required because naive T cells transferred into their original environment divide slowly.
Expansion of naive CD4+ T cells in the suitable in vivo environment does not entirely rely on the continuous subtle contacts between TCRs and self MHC/peptide complexes. To survive, adoptively transferred naive T cells require space prepared by depletion of the residual CD4+ T cell via irradiation or genetic manipulation (25). Moreover, some surface and intracellular molecules have been found differentially expressed in the naive and memory peripheral T cells, and the pools of naive and memory T cells are independently restrained by unknown homeostatic mechanisms (36, 37). Our results imply that activation of peripheral CD4+ T cell via TCR gradually diminishes their requirement for the low avidity interactions with self MHC/peptide complexes, and supports the experiments in which produced in vitro CD4+ memory T cells survived in the absence of class II MHC and Ag (7).
It is unknown why the effector CD4+ T cells that have already been activated via TCR continue to recognize self MHC/peptide ligands, but recently it became clear that several divisions of these cells are required to convert a portion of them into memory T cells (38, 39). In addition, effector CD4+ T cells can persist in vivo for the extended period of time with quantitatively and qualitatively distinct pattern of tyrosine phosphorylation (40). Our experiments indicate that the enhanced proliferation of activated CD4+ T cells occurred in the presence of specific self MHC/peptide complexes in the periphery. Therefore, the ability of effector CD4+ T cells to recognize self MHC/peptide complexes with low affinity may allow them to divide and further differentiate, even in the limited access to Ag.
An experiment in which CD4+ T cells selected by AbEp are transferred into a recipient expressing in the periphery wild-type Ab bound with many self peptides could not be performed because the majority of CD4+ T cells from AbEpIi- mice are not tolerant to wild-type peptides. However, in the lethally irradiated AbEpIi- mice reconstituted with fetal liver cells from wild-type mice, after an initial drop in CD4+ T cell number due to extensive negative selection, a pool of peripheral CD4+ T cells with TCRs encoding various Ag specificities remained unchanged for several months (41). This result indicated that some of the T cells selected by AbEp complex may cross-react with low affinity to self-derived peptides, subtly enough to avoid negative selection, but adequate to survive in the periphery.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Leszek Ignatowicz, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912-2600. E-mail address:
3 Abbreviations used in this paper: Ii, invariant chain; ß2m, ß2-microglobulin; CFSE, succinimidyl ester of carboxyfluorescein diacetate.
Received for publication September 27, 1999. Accepted for publication January 10, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
MHC- chimeras were
injected with a mixture containing equal numbers (2.5 x
106) of CFSE-labeled CD4+ lymphocytes selected
on MHC class II occupied by many peptides from B6.PL
(Thy-1.1+) and selected on single MHC II/peptide complex
from AbEpIi-ß2m-
animals (Thy-1.1-). Adoptively transferred cells could be
readily distinguished by FACS analysis on the basis of their Thy-1.1
expression. B, Contour plots depict divisions in the
compartment of cells selected on AbEp complex
(Thy-1.1-, left column) and cells selected
on Abwt (Thy-1.1+, right
column). To illustrate quantitative differences in survival of
transferred cells, data on days 2 and 16 are shown as color dot plots
using multicolor gating. Blue dots depict cells from gate
CD4+ Thy-1.1+; red dots represent events from
gate CD4+ Thy-1.1-. Results from one of six
experiments are shown. C, Total numbers of
CFSE+ and CFSE- cells in the compartment of
Thy-1.1+ and Thy-1.1- cells recovered from
peripheral lymphatic organs of animals shown in A.
Numeric value (x106) is shown in tables. Grey lines
represent number of cells transferred initially.
