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Department of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, CA 94305
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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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1:1000–10,000
T cells in primed individuals) (1, 2, 3, 4), which makes their analysis
extremely difficult. Although several methods have been developed to
study T cell responses in vivo (e.g. the adoptive transfer of T cell
clones and TCR transgenic T cells) (5, 6, 7), and some T cell responses to
particular peptide Ags (e.g. peptides from pigeon cytochrome
c) use highly restricted TCRs which enable the
tracking of Ag responses with clonotypic Abs (8, 9, 10, 11, 12), the
characterization of Ag-specific T cell responses requires a
manipulation of the normal physiology of the immune response. Thus,
although these methods have provided important insights into T cell
responses in vivo, they share some drawbacks in studies of the
conventional immune response.
T cell clones and T cells from TCR transgenic mice use a single TCR
ß heterodimer; these T cells generate a homogenous, single
affinity response to Ag. The methods used to isolate both T cell clones
and the T cells from which TCR transgenic mice are derived have used
culture conditions that are predisposed to selecting only the best
growing T cells from culture (i.e., the T cells with the proliferative
response that is "most favorable" for growth). mAbs to clonotypic
TCR - and ß-chains can be used to study T cell immune responses in
vivo, but only to those Ags that are known to elicit a clonotypic
response can be studied; moreover, the Abs will only identify T cells
with the clonotype discovered in vitro, thereby confounding the
question of whether the T cell response in vivo is similar to the T
cell response in vitro.
We have identified a novel way to investigate the response of Ag-specific T cells in vitro and in vivo that avoids these drawbacks. Here, we demonstrate a model system, using the DBA/2 response to sperm whale myoglobin (SWM)3(110–121), to study Ag-specific T cells in vitro and in vivo. Our data demonstrate that Ag-specific murine T cells up-regulate their surface expression of the CD4 molecule following Ag recognition in vitro and in vivo. The CD4high cells contain all of the Ag-specific T cell proliferative response in vivo and in vitro. The CD4high population also expresses memory/activation markers that are consistent with recently activated cells. The finding that Ag-specific T cells up-regulate CD4 expression should aid studies on the dynamics and heterogeneity of the T cell immune response in vivo, the study of suboptimal or heterogenous immune responses, and the identification of T cells that are reactive to known and unknown autoantigens.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DBA/2 mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained in the Stanford Medical Center Department of Comparative Medicine. Mice were used between 8 and 30 wk of age.
Ag proliferation assays
Groups of three to five mice were immunized at the base of the tail with 100 µl of an emulsion containing both IFA plus 10 mg/ml heat-killed Mycobacterium tuberculosis and H37RA (Difco Laboratories, Detroit, MI) plus 100 µg peptide suspended in an equal volume of Dulbecco’s PBS. The peptide SWM(110–121) (AIIHVLHSRHPG) was synthesized and HPLC-purified at the Protein and Nucleic Acid Facility (Beckman Center, Stanford University). At 8 to 10 days postimmunization, draining inguinal lymph node cells were removed, and single-cell suspensions were prepared. A total of 5 x 105 cells were incubated in 96-well flat-bottom plates in either T cell media alone or with Ag for whole lymph node proliferation assays. T cell media consisted of RPMI 1640 supplemented with 2 mM L-glutamine, penicillin/streptomycin, nonessential amino acids, sodium pyruvate, and 10 mM HEPES buffer (Life Technologies, Grand Island, NY); 50 mM 2-ME (Sigma, St. Louis, MO); and either 0.5% normal mouse serum or 5 to 10% FCS. Cells were pulsed with 1 µCi of [3H]thymidine after 72 h at 37°C with 6% CO2 and subsequently harvested after 18 h for counting on a beta plate (Wallac, Gaithersburg, MD).
FACS analysis
For studies in vitro, aliquots of lymph node cells were taken from culture, washed with FACS buffer (Dulbecco’s PBS with 2% FCS), stained for two-color flow cytometric analysis with fluorescein and phycoerythrin Abs at a predetermined optimal concentration for 20 min at 4°C, washed, and stained with propidium iodide (PI) (Sigma) just before analysis. Anti-CD45RB, anti-CD44, anti-CD69, and anti-Vß8 fluoresceinated Abs were obtained from PharMingen (San Diego, CA), as was anti-Mel-14 biotinylated Ab. Anti-CD4 and streptavidin-phycoerythrin Abs were obtained from Caltag (San Francisco, CA). A total of 1 x 105 cells were analyzed by two-color flow cytometry on a Becton Dickinson FACScan cytometer (Mountain View, CA). The data were analyzed using the Herzenberg desk facility plus Flowjo (Tree Star, San Carlos, CA) on a Power Macintosh (Apple computer, Cupertine, CA). A statistical analysis that included the two-sided Student t test was performed with Statview (Abacus Concepts, Berkeley, CA) and Excel (Microsoft, Redmond, WA). PI+ cells were excluded from analysis.
FACS sorting and proliferation assays
Single-cell suspensions were obtained either from 3-day cultures
or directly from the lymph nodes of immunized animals (harvested 8–10
days postimmunization in both cases, as described above). The cells
were stained with anti-CD4 Ab (in combination with anti-Vß8
Ab in some experiments). The cultured cells were analyzed by FACS to
determine appropriate gates, sterile-sorted into positive and negative
populations, plated into 96-well plates, pulsed with
[3H]thymidine for 18 h, and then counted on a
beta plate. Cells that had been sorted directly from immunized lymph
nodes were analyzed, and the highest 1% of cells were selected for
sorting; generally 1% analysis levels resulted in an actual sorting of
the top 0.5% of cells. The cells were cultured in 96-well plates at
varying amounts (generally between 1–2 x 104) of
CD4high vs CD4normal cells; alternately,
CD4high Vß8+ vs CD4normal
Vß8+ cells in 96-well plates containing 5 x
105 irradiated syngeneic lymph node or spleen cells with or
without Ag were used in the cultures. The cells were then cultured at
72 h before pulsing and harvesting as described above.
Limiting dilution analysis (LDA)
Cells that were obtained from culture or directly from immunized
mice, were sorted by FACS as described above. After sorting, small
aliquots of positive and negative populations were resuspended in FACS
buffer and analyzed on the FACS machine that was used for sorting to
assess for the percent purity of the sorted populations. The sorted
cells were then pelleted and titrated at varying cell numbers into
96-well plates containing irradiated DBA/2 spleen cells, 10 µM
SWM(110–121), and 10 U/ml IL-2. The plates were cultured for 10 days,
at which time the cells were transferred into fresh 96-well plates
containing irradiated DBA/2 spleen cells with or without SWM(110–121)
and without IL-2. The cells were cultured for an additional 3 days,
pulsed with 1 µCi/well [3H]thymidine, and
harvested for counting after 18 h. The proliferative response was
analyzed for responders/nonresponders using four SDs above the response
in the absence of Ag as a cutoff for positive responders. Cells were
obtained for analysis without Ag in two ways: either some wells in the
replicate wells were cultured without Ag, or some wells were split at
the time of restimulation and cultured with or without Ag. The
proliferative response of CD4high cells cultured without Ag
was consistent across experiments. In some experiments, duplicate
plates were cultured at the time of restimulation; one plate was used
for the proliferative response, while the second plate was maintained
in culture. The percent of negative wells was plotted against cells per
plate and analyzed by least-squares linearization using Cricket Graph
(Computer Associates, Islandice, NY); the exponential curve-fitting
function produced an equation of the form y = (
) x
10(z)(x). and z were derived from the data, and the
resultant precursor frequency (x) was calculated by setting y =
0.37 according to Poisson statistics.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The DBA/2 response to SWM(110–121) that had been previously
characterized in our lab (4, 13, 14, 15) was initially used to analyze
Ag-reactive T cells. Groups of mice were immunized with peptide, and
immune lymph node cells were harvested 8 to 10 days later and cultured
with or without the immunizing Ag. Periodically, aliquots of cells were
taken from culture, stained with Abs to cell surface molecules, and
analyzed by flow cytometry for kinetics of expression by live
(PI-) cells (Fig. 1
).
At 48 h, and more obviously at 72 and 96 h, a subpopulation
of cells demonstrated increased CD4 expression (Fig. 1). These
CD4high cells were also CD45RBlow,
CD62Llow, CD69high, and CD44high
(Fig. 1). T cells from immunized lymph nodes cultured for similar
periods of time without Ag showed no comparable CD4high
subpopulation (data not shown). The percentage of CD4high
cells increased steadily throughout the culture period, from <0.4% of
live cells at time 0 to 10 to 20% of cells by 96 h (Fig. 1). The
increased percentage was due both to the death of other cells in the
culture over time and to an absolute increase in the
CD4high cell numbers (data not shown). A statistical
analysis of the CD4high levels of 13 separate experiments
at 72 h postimmunization (the usual time of pulsing in
proliferation assays) showed that the difference in CD4 expression
between CD4high and CD4normal was highly
significant (unpaired Student’s t test, p
< 0.00001). While there was some variance in the absolute level of CD4
expression in the experiments, the ratio of the CD4high to
CD4normal was remarkably consistent over all experiments at
3.5:1. The mean ratios of CD45RB and CD44 in the CD4high
population were also significantly different from the
CD4normal population. The CD45RB expression in the
CD4high group was approximately one-half of the
CD4normal group. The CD4normal population
showed two subpopulations; one expressed 165-fold less CD44 than the
CD4high cells, while the other expressed 3-fold less
CD44 than the CD4high population (Fig. 1).
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) were used to
establish sorting gates. When the cells were sorted and pulsed with
[3H]thymidine, the CD4high population
contained all of the proliferating cells from the whole lymph node
preparation, while the CD4normal cells did not proliferate
above background (Fig. 2B). The whole lymph
node cell response to Ag from the same culture was used as a control
(Fig. 2A). Thus, all of the Ag-specific proliferative
response seen in vitro in response to SWM(110–121) was contained in
the CD4high cell population.
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It was possible that only a small percentage of the
CD4high cells was actually proliferating in the
sorting experiment shown in Figure 2. Thus, we used LDA to quantify the
enrichment of Ag reactivity in the CD4high population. SWM
immune DBA/2 lymph node cells were cultured with Ag for 3 days and
sorted into CD4high and CD4normal
subpopulations and then plated at titrated numbers onto irradiated
DBA/2 spleen cells in the presence of Ag and IL-2. After 10 days of
culture, the cells were transferred to plates with fresh Ag and APCs in
the absence of IL-2 and pulsed for a proliferative response after
another 3 days of culture. The fraction of negative wells was
calculated, allowing for the determination of Ag-specific T cell
frequency according to Poisson statistics (Fig. 3). The results presented in Figure 3
demonstrate that the CD4high subpopulation from the culture
contained a substantial enrichment of the Ag-responsive cells; 1 in 5.1
CD4high cells were Ag-reactive compared with typical
estimates of Ag frequency in immunized lymph nodes from 1:1000 to
1:10,000 whole lymph node cells (1, 2, 3) and compared with the estimate
of whole SWM-reactive T cells in primed DBA/2 mice of 1:11,625 that was
published previously by our lab (4). The mean of five LDA experiments
indicated that 1 in 8.2 CD4high cells was Ag-specific.
While the CD4normal curve was not entirely horizontal (Fig. 3), the CD4normal cells showed no Ag reactivity after
pulsing directly after harvesting from the immunized lymph node (Fig. 2B) or after restimulation with Ag for 3 days before
pulsing (Fig. 4). A post-sort analysis
established that the residual reactivity seen in the LDA experiments
was due to a minor population (<1.5%) of CD4high cells
that contaminated the CD4normal group during FACS sorting
(data not shown).
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These results demonstrated that the CD4high cells
contained Ag-responsive cells in vitro. An adaptation of this method to
studies in vivo was then attempted. FACS analysis of freshly harvested
lymph node cells from DBA/2 mice (taken at various time points after
immunization with SWM(110–121)) showed no obvious expansion of
CD4high cells (see Fig. 5A, top panel,
compared with Fig. 1). We reasoned that the Ag-reactive
CD4high T cell population was present but had not expanded
as dramatically in vivo as in vitro. To test this hypothesis, we again
used the DBA/2 response to SWM(110–121), which shows a predominant
Vß8 response when cells are cloned and T cell hybridomas are made
(13, 14, 15). We sorted CD4high Vß8+ T cells
directly from the lymph nodes of SWM-immunized DBA/2 mice; gates set to
select the highest CD4 expressing Vß8+T cells
(representing 0.5% of lymph node cells) were used, and those cells,
vs CD4normal Vß8+ T cells, were cultured with
naive, irradiated DBA/2 spleen cells with or without Ag (the lymph node
T cell proliferative response from the same mice served as control)
(Fig. 4). The CD4high Vß8+ T cells
demonstrated a robust Ag proliferative response, while the
CD4normal Vß8+ T cells did not. These
experiments demonstrated that the Ag-reactive T cell
CD4high subpopulation could be identified in vivo.
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Next, we quantified the enrichment of the CD4high
Ag-specific T cell precursor frequency of CD4high T cells
activated in vivo using LDA. We sorted CD4high
Vß8+ T cells directly from freshly harvested
SWM(110–121)-immunized lymph nodes at 8 days postimmunization (Fig. 5A); the top 1% of CD4 brightness in the
CD4+ Vß8+ cell population was used as a
sorting gate (Fig. 5A). We cultured titrated, sorted
cell numbers in 96-well plates with naive, irradiated DBA/2 spleen
cells, Ag, and IL-2, and restimulated the cultures at 10 days. We
consistently found a substantial enrichment of Ag reactivity in the
Vß8+ response (1 in 32.4 CD4high cells, Fig. 5B) in the 8 day in vivo SWM(110–121) response. The
result was quite consistent over five LDA experiments, with a mean
indicating that 1/32.8 CD4high cells was Ag-specific from
primed lymph nodes in vivo.
The frequency of CD4high Vß8+ cells for
SWM(110–121) in vivo was lower than that seen in the in vitro analysis
(Fig. 3). The lower precursor frequency demonstrated in vivo compared
with studies in vitro could be attributed in part to the technical
difficulty of extracting cells directly from the mouse and
sorting/culturing them for prolonged periods. In addition, an analysis
of the response of the CD4high T cells to purified protein
derivative demonstrated that some CD4high cells, when taken
directly from immunized mice, were primed by the purified protein
derivative contained in the CFA that was used as adjuvant (data not
shown). CFA-primed cells do not expand during culture with SWM; hence,
the CD4high population in vitro is further enriched for
SWM-reactive cells at the time of cell sorting from culture. While the
CFA primed cells do not expand during restimulation in vitro with SWM,
they will dilute the frequency of CD4high cells that
recognize SWM at the time of sorting (sorting is performed before
expansion with the in vivo-primed cells). A final explanation of the
fourfold less enrichment in vivo compared with the in vitro sorting of
CD4high is suggested by a comparison of the empiric sort
gates chosen in vivo (Fig. 5A) with the gates
established in vitro (Fig. 1). The consistent mean ratio of
CD4high to CD4normal expression in vitro
suggested that the gates chosen in vivo allowed a significant number of
CD4normal cells into the "CD4high"
population.
We have demonstrated that T cells from all mouse strains studied to date (following immunization with peptides or proteins) up-regulate CD4 expression in response to Ag; e.g., PLJ x SJL)F1 mice in response to myelin basic protein (unpublished observations), nonobese diabetic mice in response to self and foreign peptides, and naive hen egg lysozyme(46–61)-responsive transgenic T cells in culture (our unpublished observations). We postulate that the up-regulation of CD4 in response to peptide Ags is a universal T cell response to initial confrontation with "nominal" Ag in vivo. CD4high T cells contain a heterogenous population of responding T cells.
Next, we investigated whether CD4high expression could be
used as a method of identifying and characterizing those Ag-specific
cells in vivo that are not normally expanded under competitive culture
conditions (i.e., suboptimal T cell proliferative responses). The
CD4high approach allowed for the possibility of
individually expanding suboptimally reactive T cells directly from the
immunized lymph node. To accomplish this expansion, LDAs were conducted
as described above, and CD4high Vß8+ cells
were plated into replicate plates at the time of restimulation. One of
the two plates was then cultured for 3 additional days and pulsed with
[3H]thymidine to establish the dilution of cells at which
one cell or less was present in the well. The proliferative responses
of individual positive wells grown out from initial conditions at 1
cell/well (Fig. 6) (as calculated by the
LDA from Poisson statistics) were plotted. The responses of positive
wells expanded under identical culture conditions from individual
CD4high Vß8+ cells showed a continuous
distribution (over 3 logs of cpm) of proliferative responses from
very high responders to very low responders (Fig. 6). This distribution
stands in contrast to the expected behavior of a clonal population,
which should show a normal distribution of proliferative responses
(i.e., most of the response occurring near a mean, with some outliers
on each end). The uncloned CD4high cells from immune lymph
nodes, on the contrary, show equal numbers of cells proliferating at
every level (Fig. 6). It is natural to speculate that if the
fast-growing CD4high cells (as plotted in Fig. 6, far
right) were cultured under standard cloning conditions with
slow-growing CD4high T cells (Fig. 6, left), the fast cells would outgrow the slower cells.
The advantage of this limiting dilution method of cloning cells
directly from the lymph node is that the separate cells, when cultured
at one cell (or less) per well, can be expanded without the pressure of
competitive growth, thus enabling a study of suboptimal responders to
Ag in vivo. There are two likely explanations for the unequal growth of
responding CD4high cells plated under identical conditions.
One possibility is that an unknown stimulus continuously distributed
under the conditions of culture causes T cells with inherently equal Ag
responsiveness to proliferate unequally. The second explanation is that
the responding T cells display a continuous distribution of growth
(proliferation) in response to a single Ag concentration. If the growth
response reflects an intrinsic difference between the responding T
cells, such as TCR affinity for Ag, the phenotype of low vs high
responders should persist after repetitive stimulation and rest of the
T cells. The system outlined here allowed us to investigate that
question. Preliminary results indicate that low responder cells can be
cloned (from the replicate plates) and maintained in culture, and not
only retain their low proliferative phenotype but also express
different (nondominant) Vß repertoires from the high responder cells
(M.F., manuscript in preparation).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and 4). Moreover, depleting the
CD4high T cells from whole lymph node populations that are
responsive to the immunizing Ag ablates the immune response (i.e., the
CD4normal population that remains after depleting the
CD4high T cells does not proliferate when pulsed (Fig. 2B), nor does it proliferate when cultured with the
immunizing Ag (Fig. 4)). An LDA to obtain the precursor frequency of
Ag-specific T cells in the CD4high populations both in
vitro and in vivo (Figs. 3 and 5) demonstrates a substantial
(100–1000-fold) enrichment of Ag-specific T cells compared with
traditional cloning methods in general (1, 2, 3, 5, 16) and with our
previous results in the SWM/DBA2 system in particular (4). In addition,
the CD4high marker can be used to follow the Ag-specific T
cell response in vivo (Fig. 5) and allows a method to study T cells
that have a suboptimal response to Ag (Fig. 6). The possibility of
studying a variety of T cells with different Ag recognition properties
while holding the Ag dose constant is a novel approach that bypasses
some of the obstacles presented by the traditional method of studying a
single, high affinity T cell in response to a varying Ag dose. Since we
have demonstrated the up-regulation of CD4 by T cells upon first
contact with their nominal Ag in several different Ag systems and in
several strains of mice, these findings should have broad applicability
in immunology. These results introduce the possibility of using the
CD4high marker to study a variety of Ag-specific autoimmune
T cell responses in vitro and in vivo. For example the study of
suboptimal T cell responses to date has used T cell clones that respond
well to their nominal Ag but respond suboptimally to different doses of
an altered peptide ligand (17, 18, 19). The use of the CD4high
marker allows us to invert this approach; using a constant dose of an
Ag, we can derive T cells that respond suboptimally to that Ag in vivo.
It has now been well-established that low proliferative responder T
cells can manifest Ag responses to altered peptides by alterations in
cytokine production (17, 18, 19). We anticipate that T cells with
suboptimal T cell responses might demonstrate an altered cytokine
profile compared with T cells that respond with the "usual" high
proliferative response to Ag. As another potential application, since
the expression of CD4 increases in T cells following their recognition
of Ag, and the CD4high T cells can be recovered directly
from mice, this method can potentially be applied to isolating
autoreactive T cells and identifying the autoantigen recognized by the
CD4high autoreactive T cells (20).
It is somewhat surprising that CD4 up-regulation by T cells in response
to their nominal Ag has not been previously reported. Many of the
earlier studies of T cell activation used Jurkat cells or T cell
hybridomas and stimulated them with less-specific reagents, including
lectins, ionomycin, and Abs; in some of these experiments, CD4
reportedly decreased in expression following activation (21, 22, 23). In
our own lab, we have observed that PMA and ionomycin stimulation can
cause an apparent down-regulation of CD4 (our unpublished data). The
different outcome of CD4 expression following stimulation with specific
Ag vs "stronger" or less-specific T cell-activating agents may
reflect a physiologic difference in the mechanism of activation.
Studies using T cell clones have also not shown CD4 up-regulation. We
derived Ag-specific T cell lines and examined CD4 expression after 10
days of culture; the surviving cells were CD4high as
expected from Figure 1 (data not shown). We then compared the CD4
levels of an established T cell clone (11.3, derived from DBA/2 mice by
immunization with SWM(110–121) followed by alternating rest and
activation) at 3 days and at 10 to 14 days following activation; the
CD4 levels did not change regardless of the activation state (data not
shown). From these experiments, we conclude that T cell clones have
already up-regulated CD4 expression, which explains why CD4
up-regulation was not seen in studies involving T cell clones. These
results also suggest that CD4high may be a persistent
phenotype marking memory-like cells; this possibility is supported by
LDA experiments showing Ag-specific cells in the CD4high
group in vivo for 
4 wk postimmunization (M.F., manuscript in
preparation).
Although it was not studied here, it is tempting to speculate upon the biologic significance of CD4 up-regulation in the T cell response to Ag. The role of CD4 as a coreceptor has been persuasively argued by Janeway, who proposed that the association of CD4 with the TCR during the process of T cell activation could allow enhanced signal amplification (24, 25). In contrast, naive T cells showed no association of CD4 with the TCR (26). In the CD4 coreceptor model, the increased CD4 expression we have demonstrated could provide a mechanism for enhanced signal transduction via a stoichiometric increase in the amount of CD4 in the TCR activation complex. Another view of the role of CD4 in TCR signaling has been proposed by Germain and coworkers (27), using a density model of partial agonist signaling. Germain suggested that low vs high densities of CD4 could affect the dissociation time of the TCR with its peptide/MHC complex (27). In the density model, the higher CD4 surface expression that we have demonstrated on activated Ag-specific T cells could enhance signaling capability via an enhancement of overall T cell:MHC/peptide avidity. These and other models of the effect of increased CD4 expression on Ag-specific, activated T cells may be amenable to experimentation using the approach described in this paper.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. C.Garrison Fathman, Stanford University School of Medicine, Department of Medicine, Division of Immunology and Rheumatology, Room S021, Stanford, CA 94305. E-mail address:
3 Abbreviations used in this paper: SWM, sperm whale myoglobin; LDA, limiting dilution analysis; PI, propidium iodide.
Received for publication August 4, 1997. Accepted for publication March 23, 1998.
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 A. G. Schrum and L. A. Turka The Proliferative Capacity of Individual Naive CD4+T Cells Is Amplified by Prolonged T Cell Antigen Receptor Triggering J. Exp. Med., September 16, 2002; 196(6): 793 - 803. [Abstract] [Full Text] [PDF]
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 H. Reijonen, E. J. Novak, S. Kochik, A. Heninger, A. W. Liu, W. W. Kwok, and G. T. Nepom Detection of GAD65-Specific T-Cells by Major Histocompatibility Complex Class II Tetramers in Type 1 Diabetic Patients and At-Risk Subjects Diabetes, May 1, 2002; 51(5): 1375 - 1382. [Abstract] [Full Text] [PDF]
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 E. J. Novak, S. A. Masewicz, A. W. Liu, A. Lernmark, W. W. Kwok, and G. T. Nepom Activated human epitope-specific T cells identified by class II tetramers reside within a CD4high, proliferating subset Int. Immunol., June 1, 2001; 13(6): 799 - 806. [Abstract] [Full Text] [PDF]
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L. M. Ebert and S. R. McColl Coregulation of CXC Chemokine Receptor and CD4 Expression on T Lymphocytes During Allogeneic Activation J. Immunol., April 15, 2001; 166(8): 4870 - 4878. [Abstract] [Full Text] [PDF]
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M. Fasso, N. Anandasabapathy, F. Crawford, J. Kappler, C. G. Fathman, and W. M. Ridgway T Cell Receptor (Tcr)-Mediated Repertoire Selection and Loss of Tcr V{beta} Diversity during the Initiation of a Cd4+ T Cell Response in Vivo J. Exp. Med., December 18, 2000; 192(12): 1719 - 1730. [Abstract] [Full Text] [PDF]
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R. A. O'Connor, J. S. Jenson, and E. Devaney NO Contributes to Proliferative Suppression in a Murine Model of Filariasis Infect. Immun., November 1, 2000; 68(11): 6101 - 6107. [Abstract] [Full Text] [PDF]
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C. G. Radu, S. M. Anderton, M. Firan, D. C. Wraith, and E. S. Ward Detection of autoreactive T cells in H-2u mice using peptide-MHC multimers Int. Immunol., November 1, 2000; 12(11): 1553 - 1560. [Abstract] [Full Text] [PDF]
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 S. Sarafova and G. Siu Precise arrangement of factor-binding sites is required for murine CD4 promoter function Nucleic Acids Res., July 15, 2000; 28(14): 2664 - 2671. [Abstract] [Full Text] [PDF]
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G. J. Wiegers, I. E. M. Stec, W. E. F. Klinkert, and J. M. H. M. Reul Glucocorticoids Regulate TCR-Induced Elevation of CD4: Functional Implications J. Immunol., June 15, 2000; 164(12): 6213 - 6220. [Abstract] [Full Text] [PDF]
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H. Skrenta, Y. Yang, S. Pestka, and C. G. Fathman Ligand-Independent Down-Regulation of IFN-{gamma} Receptor 1 Following TCR Engagement J. Immunol., April 1, 2000; 164(7): 3506 - 3511. [Abstract] [Full Text] [PDF]
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G. L. Costa, J. M. Benson, C. M. Seroogy, P. Achacoso, C. G. Fathman, and G. P. Nolan Targeting Rare Populations of Murine Antigen-Specific T Lymphocytes by Retroviral Transduction for Potential Application in Gene Therapy for Autoimmune Disease J. Immunol., April 1, 2000; 164(7): 3581 - 3590. [Abstract] [Full Text] [PDF]
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N. Manjunath, P. Shankar, B. Stockton, P. D. Dubey, J. Lieberman, and U. H. von Andrian A transgenic mouse model to analyze CD8+ effector T cell differentiation in vivo PNAS, November 23, 1999; 96(24): 13932 - 13937. [Abstract] [Full Text] [PDF]
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K. Lejon C. and G. Fathman Isolation of Self Antigen-Reactive Cells from Inflamed Islets of Nonobese Diabetic Mice Using CD4high Expression as a Marker J. Immunol., November 15, 1999; 163(10): 5708 - 5714. [Abstract] [Full Text] [PDF]
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M. Ahmadzadeh, S. F. Hussain, and D. L. Farber Effector CD4 T Cells Are Biochemically Distinct from the Memory Subset: Evidence for Long-Term Persistence of Effectors In Vivo J. Immunol., September 15, 1999; 163(6): 3053 - 3063. [Abstract] [Full Text] [PDF]
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J. T. Wise, T. J. Baginski, and J. L. Mobley An Adoptive Transfer Model of Allergic Lung Inflammation in Mice Is Mediated by CD4+CD62LlowCD25+ T Cells J. Immunol., May 1, 1999; 162(9): 5592 - 5600. [Abstract] [Full Text] [PDF]
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C.-P. Liu, K. Jiang, C.-H. Wu, W.-H. Lee, and W.-J. Lin Detection of glutamic acid decarboxylase-activated T cells with I-Ag7 tetramers PNAS, December 19, 2000; 97(26): 14596 - 14601. [Abstract] [Full Text] [PDF]
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0.5% of lymph node cells) or
CD4normal Vß8+ cells, which were then
cultured for 3 days with irradiated DBA/2 spleen cells plus
SWM(110–121) before pulsing with [3H]thymidine. Error
bars represent the SEM; one representative experiment of at least three
is shown.
