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*
Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104;
Basel Institute for Immunology, Basel, Switzerland; and
Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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F1 MHC mismatch in which only donor
cells respond. Recipients were sacrificed at serial time points to
assess engraftment efficiency, and the extent of donor cell activation
and proliferation. These data were used to calculate alloreactive T
cell frequencies that varied 30-fold (0.71 ± 0.31% to 21.05
± 3.62%), depending upon whether it was assumed that all donor cells
injected became established and were capable of responding, or that
only those present at later time points (24–72 h) were available to
respond. By measuring the number of cells established in the recipient
24 h after transfer, before proliferation, we calculated an in
vivo alloreactive frequency of 
7%. Using CD69 expression at 48
h to quantify activation, we found that 40–50% of the alloactivated
CD4+ donor T cells do not divide. Studies of cotransferred
congenic and allogeneic cells demonstrated that bystander proliferation
does not occur. We conclude that accurate calculations of alloreactive
precursor frequency must account for both proliferation and cell
engraftment. When this is done, a high percentage of alloreactive T
cells exists across an MHC mismatch, but not all alloreactive cells
proliferate in vivo. Bystander proliferation is negligible, revealing
exquisite specificity to the alloresponse. These data provide a novel
approach to quantify alloreactive T cell responses during specific
immunomodulatory strategies in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The broad range of estimated alloreactive T cell precursor frequencies results in part from the lack of standard and reliable experimental methods to address this problem. One commonly employed method is limiting dilution analysis (LDA),4 a technique that uses the statistical probability of cells cultured individually to grow or to develop effector function (5, 8, 9, 10, 11, 12, 13). However, in the case of CD8+ T cells, the number of precursor T cells detected by LDA has been shown to be 50–500 times lower than that detected by other methods, such as MHC tetramer staining (14). Whether or not similar limitations apply to the LDA technique in the case of CD4+ T cells has not been determined, due to the difficulties at present in the development and use of MHC class II tetramers.
More recently, enzyme-linked immunospot (ELISPOT) has been used to estimate the alloreactive T cell precursor frequency. This sensitive method of detecting cytokine secretion has yielded precursor frequencies of 1/4,000 to 1/20,000 (15). ELISPOT may also underestimate the true precursor frequency by ignoring alloreactive cells that do not elaborate the particular cytokine detected at the time the assay is performed. In addition, ELISPOT cannot distinguish cells that are activated through their TCR binding to Ag plus MHC from cells that do not bind alloantigen, but respond to soluble factors secreted by neighboring cells.
To study alloreactive T cells under more physiologic conditions, and consequently better estimate the precursor frequency, we used the fluorescent cytoplasmic dye CFSE in combination with flow cytometry. This dye’s property of distributing equally to daughter cells upon division has enabled it to become a highly useful tool to study lymphocyte division kinetics and differentiation in a variety of in vitro and in vivo systems (16, 17). We report in this study quantitative methods to estimate the alloreactive T cell frequency in an in vivo system based on CFSE dilution profiles. We also quantitatively define the in vivo activation of adoptively transferred allogeneic T cells as a function of division cycle, and use this to give a direct evaluation of allogeneic bystander activation in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 (H-2b), C57BL/6 Thy-1.1
(C57BL/6J-Igha-Thy-1a-Gpi1a,
H-2b), bm12
(B6.C-H2bm12/KhEg), and C57BL6xDBA
F1/J (B6D2F1,
H-2b/d) mice were purchased from The Jackson
Laboratory (Bar Harbor, ME). BALB/c Thy-1.1 mice were provided by R.
Dutton (Saranac, NY), and anti-bm12 TCR-transgenic B6 mice
(anti-bm12) were previously described (18). The
transgenic TCR expressed by these mice uses V
2 and V
8, and >95%
of peripheral T cells are
V2+V8+ (data not
shown). All mice were housed in a pathogen-free facility and were used
at age 8–16 wk.
Abs and CFSE labeling
Spleen and lymph nodes from donor mice were harvested, and
single cell suspensions were prepared and labeled with CFSE (Molecular
Probes, Eugene, OR), as previously described (16).
Fluorochrome-labeled mAbs against CD4, CD8, CD44, CD45RB, CD62L, CD69,
Thy-1.1, Thy-1.2, CD16/CD32 (Fc block), and V8, biotin-conjugated
V2, fluorochrome-labeled streptavidin, and isotype controls were
purchased from PharMingen (San Diego, CA).
Adoptive transfer of alloreactive cells
A total of 3–5 x 107 CFSE-labeled C57BL/6 Thy-1.1 splenocytes and lymph node cells were injected i.v. into B6D2F1 mice in a total volume of 0.25 ml sterile PBS. Syngeneic C57BL/6 (Thy-1.2) mice were used as recipients in control experiments. Recipients were sacrificed at 24, 48, or 72 h, and spleen and lymph nodes (superficial inguinal, axillary, lateral axillary, cervical, and paraaortic) were harvested. For studies of bystander activation, CFSE-labeled B6D2F1 splenocytes and lymph node cells (2.5 x 107) were coadoptively transferred with unlabeled C57BL/6 Thy-1.1 splenocytes and lymph node cells (2.5 x 107) into B6D2F1 mice. Recipients were sacrificed at 48 h. For bm12 studies, CFSE-labeled anti-bm12 splenocytes and lymph node cells (5 x 106) were coadoptively transferred with CFSE-labeled bm12 splenocytes and lymph node cells (5 x 106) into bm12 mice. Recipients were sacrificed at 24 h.
In vitro MLRs
MLRs were established in 24-well flat-bottom plates between CFSE-labeled BALB/c-Thy-1.1 spleen and lymph node cells (2.5 x 106/well) and irradiated C57BL/6 (Thy-1.2) splenocytes (7.5 x 106/well). In control experiments, BALB/c-Thy-1.1 splenocytes were used as stimulator cells. Cells were harvested at 24, 48, 72, and 96 h; stained for CD69, CD4, Thy-1.1, and TOPRO-3 (16); and analyzed by flow cytometry.
Flow cytometry
Cells were washed in PBS containing 2% FCS and 0.02% azide at
4°C. Unlabeled Ab against CD16/CD32 (anti-FcR
III/FcRII) was
used to block FcR binding. Between 1 and 5 x
106 cells were stained with either PE-conjugated
anti-Thy-1.1 or -Thy-1.2, PerCP-conjugated anti-CD4, and either
biotinylated anti-CD69, -CD44, -CD45RB, or -CD62L, followed by
APC-conjugated streptavidin. FITC-conjugated anti-CD4 and
PE-conjugated anti-CD8 were used to determine the percentage of T
cells in donor splenocytes and lymph node cells. In experiments using
CFSE-labeled bm12 and anti-bm12 cells, harvested cells were stained
with biotinylated anti-V2, PE-conjugated anti-V8, and
APC-conjugated anti-Thy-1.2, followed by CyChrome-conjugated
streptavidin. Four-color flow cytometry was performed on a FACScalibur
(Becton Dickinson Immunocytometry Systems, San Jose, CA), and cells
were analyzed using CellQuest acquisition and analysis software. A gate
defining lymphocytes by forward and side scatter properties was used in
all analyses. Between 0.5 and 2 x 106 total
events were collected yielding 1–5 x 103
Thy-1.1 events.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, CFSE was used to assess the in vivo proliferative
kinetics of parental T cells transferred to an F1
hybrid recipient. C57BL/6 (H-2b) splenocytes and
lymph node cells transferred i.v. to B6D2F1
(H-2b/d) recipients are potentially alloreactive
against recipient Ags contributed by the nondonor haplotype parent. For
these analyses, we used C57BL/6 donors congenic for the Thy-1.1 allele
and B6D2F1 recipients that carried the Thy-1.2
allele. Therefore, we were able to use an anti-Thy-1.1 Ab to detect
the donor cells harvested from recipient peripheral lymphoid organs
after adoptive transfer. A gate defining the CD4 subset was used to
distinguish this population (Fig. 1
, A and B).
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, D and E). Heterogeneity in the
alloreactive proliferative response was seen, as some T cells had
divided as many as eight times by 72 h, whereas other donor cells
had divided only once or not at all. In contrast, no proliferation was
detected when C57BL/6 Thy-1.1 splenocytes and lymph node cells were
transferred into syngeneic C57BL/6 Thy-1.2 mice, demonstrating
negligible background proliferation in this system (Fig. 1C). Finally, based on the proliferative profile, the
majority of the recovered donor cells consisted of the CD4 T cell
subset (Fig. 1E). The difference in the total cell counts in
Fig. 1D and the CD4 cell counts in Fig. 1E is
presumably accounted for by the CD8 T cells. Development of methods to quantitatively assess allospecific T cell proliferation in vivo
The number of cells within each CFSE fluorescent peak is determined by analysis of the flow-cytometric histogram plots. From the number of events comprising a peak, representing alloreactive daughter cells with identical proliferative history, the number of precursors that divided to generate that population can be extrapolated by dividing by 2n, in which n is the division cycle number (16). For example, if 100 events are counted in the third peak from the right, representing two rounds of cell division (n = 2), the number of precursor alloreactive cells that generated these 100 cells equals 100/22 or 25 cells. Summing the extrapolated precursors for division cycles 1 to n yields the size of the alloreactive precursor pool recovered from the recipient and analyzed by flow cytometry that divided in response to alloantigen (Pdiv).
The frequency of alloreactive T cells is expressed as the ratio of two
quantities: the number of dividing cells
(Pdiv), which makes up the numerator,
and the total number of cells (Ptot),
comprising the denominator (Fig. 2).
Pdiv is relatively straightforward to
estimate, as described above. However,
Ptot is complex and its estimate has
contributed to the limitations and variability of previous attempts to
estimate the alloreactive precursor frequency. We used a single method
to determine Pdiv in conjunction with
three distinct methods to determine
Ptot in estimating the alloreactive T
cell precursor frequency in vivo. As depicted in Fig. 2, the methods
are distinguished by the time point used to determine
Ptot: after adoptive transfer at the
time of maximal proliferation (method 1), before adoptive transfer
(method 2), and after adoptive transfer but before cell division
(method 3). Therefore, the value of
Ptot determined by each of the three
methods described determines the estimated value of the alloreactive T
cell frequency.
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Method 2 attempts to define the alloreactive T cell precursor pool size as a portion of the total number of donor T cells transferred. In this method, Pdiv is divided by the number of transferred donor T cells (Ptot2), yielding the ratio of Pdiv to Ptot2 we define as the precursor frequency. This approach to estimating Ptot is limited by the fact that by 72 h a portion of the transferred cells will be in recipient sites that we did not sample (liver, lungs, etc.), thereby excluding their responders from analysis and underestimating the precursor frequency. Furthermore, even in the sites we examined, many cells may not become established due to lack of physical space or functional niches. This also will bias the analysis toward an underestimate.
In method 3, we attempt to more accurately define the potentially alloreactive T cell pool by measuring the size of the donor T cell pool 24 h after adoptive transfer, before any proliferation has occurred. The alloreactive T cell frequency is determined by dividing Pdiv by the number of donor T cells recovered at 24 h from a recipient spleen and lymph nodes (Ptot3). We term the resulting number the recovered precursor frequency. Recognizing that not all transferred lymphocytes will have homed to the secondary lymphoid tissues, we reasoned that this method most accurately quantifies the alloreactive T cell frequency, because it is not biased by an abundance of proliferative cells, and it reflects the donor cells that found niches in the lymphoid sites sampled before their onset of proliferation.
Results of three methods to quantitatively assess allospecific T cell activation and proliferation in vivo
The responder frequency (method 1) of B6
(H-2b) splenocytes and lymph node cells reacting
to H-2d Ags in B6D2F1 hosts
(H-2b/d), was 21.05 ± 3.62% and 26.39
± 3.72% for total T cells and CD4 T cells, respectively
(n = 7, Table I). The
precursor frequency (method 2) was calculated to be 0.71 ± 0.31%
of all donor T cells and 0.58 ± 0.21% of CD4 T cells
(n = 7, Table I). The recovered precursor frequency
(method 3) was determined to be 6.84 ± 3.90% for all T cells,
and 5.71 ± 3.39% for CD4 T cells (n = 3, Table I). Of note, in each experiment Ptot3
was <10% of Ptot2. This low recovery
confirmed our suspicions that not all transferred cells would traffic
to and engraft in the lymphoid organs that we sampled. Therefore,
method 3 may give the most accurate estimate of the alloreactive T cell
frequency compared with methods 1 and 2. By each method, the
alloreactive T cell frequency was not detectable for the syngeneic
control because no proliferation was observed (Fig. 1C).
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An assumption of method 3 is that among injected cells, the
alloreactive cells do not preferentially localize to the recipient
spleen and lymph nodes. If they did, method 3 would overestimate the
alloreactive T cell precursor frequency. To directly test this, we
coadoptively transferred into bm12 recipient mice a 1:1 mixture of
CFSE-labeled spleen and lymph node cells from bm12 mice and from
TCR-transgenic mice specific for the class II bm12 alloantigen. In this
system, 50% of the transferred cells are syngeneic to the host and
therefore not reactive and 50% are reactive to recipient alloantigen.
After 24 h, the recipients were sacrificed and their spleen and
lymph node cells were harvested and analyzed by flow cytometry to
assess syngeneic vs allogeneic donor cell recovery in the host
secondary lymphoid organs. T cells from anti-bm12 mice are >95%
V2+V8+, whereas
2% of T cells from bm12 mice use this TCR heterodimer (data
not shown). For this analysis, allogeneic anti-bm12 T cells were
defined as CFSE+ Thy-1.2+
V2+V8+ cells, while
syngeneic donor bm12 T cells were defined as
CFSE+ Thy-1.2+ cells that
were V2 and/or V8 negative. Because 2% of normal bm12 T cells
are V2+V8+, we
adjusted the final values to account for the bm12 cells that bear this
TCR heterodimer but are not alloreactive. With this analysis, we found
that 42 ± 2.12% (n = 2) of the recovered donor
cells were anti-bm12, a value similar to the original 50%
proportion transferred. This suggests that alloreactive and
nonalloreactive T cells exhibit similar homing to recipient secondary
lymphoid organs in this model. Therefore, we believe that our
yield-based calculations (methods 1 and 3) are not biased significantly
by differential homing.
Comparison of in vivo and in vitro MLR
We wished to compare the results of the in vivo quantitative methods with those obtained from an in vitro alloreaction. In vitro MLRs were established between irradiated C57BL/6 Thy-1.2 stimulator splenocytes and CFSE-labeled BALB/c Thy-1.1 responder spleen and lymph node cells. The division kinetics of the BALB/c lymphocytes in vitro were identical with those of the C57BL/6 spleen and lymph node cells in vivo. In these experiments, the alloreactive T cell precursor frequency was calculated using a yield-independent approach similar to that described in method 2 above. This is the valid method because the starting number of potentially alloreactive responder cells in each experiment is known (2.5 x 106), and the proliferative history of all allogeneic precursor cells, including cells that have died (16), is visualized by the CFSE-fluorescent profile of the daughter cells generated by the time of harvest from their reaction wells (72 h). In this in vitro system, the frequency of BALB/c (H-2d) splenocytes and lymph node cells reacting to B6 (H-2b) stimulator cells was 4.61 ± 2.22% for all T cells (n = 6) and 1.82 ± 0.95% for CD4 T cells (n = 4). These results are similar to those calculated for the adoptive transfer experiments using method 3.
Division cycle-associated T cell activation marker expression in vivo
Based on our earlier findings showing that when splenocytes are stimulated in vitro with anti-CD3 and anti-CD28 (16), and when mice adoptively transferred with DO11.10 T cells that bear a transgenic TCR specific to OVA peptide are immunized (17), not all T cells proliferate, we reasoned that in this system all alloreactive T cells might not divide. In the DO11.10 system, the Ag-specific transgenic T cells can be identified using an anti-clonotypic Ab, but no such marker exists to distinguish heterogeneous alloantigen-specific T cells. However, because we demonstrated that nearly 100% of mitogen-stimulated splenocytes in vitro and clonotype-positive cells recovered from immunized mice express the surface markers of activation CD25 or CD69, we reasoned that adoptively transferred allogeneic T cells that bind alloantigen can be distinguished by their expression of surface markers of activation or differentiation. To this end, we examined the expression of CD44, CD45RB, CD62L, and CD69.
Naive T cells express on their surface a low density of CD44 and a high
density of CD45RB and CD62L, and after encountering Ag, a
CD44highCD45RBlowCD62Llow
phenotype is associated with T cell effector function (19, 20). We tracked the expression of CD44, CD45RB, and CD62L on
individual adoptively transferred allogeneic T cells at 72 h. At
the single-cell level, the expression of these differentiation markers
was associated with cell division in a manner identical with that seen
with adoptively transferred TCR-transgenic T cells (17, 21). CD44 was up-regulated after a single round of cell division
and remained elevated throughout the eight mitoses visualized by CFSE
(Fig. 3), CD45RB expression decreased
2-fold with each division, and the surface expression of CD62L was
down-modulated by division cycles 7 and 8 after undergoing a triphasic
expression response during eight rounds of cell division (data not
shown).
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, C, D, and
E), before adoptive transfer, a subpopulation of donor T
cells already expressed high levels of CD44 (data not shown),
indicating the presence of activated or memory T cells raised to
environmental Ags. Following adoptive transfer, but before cell
division began (24 h), this subpopulation of CD44-expressing donor T
cells remained detectable in both allogeneic and syngeneic settings
(Fig. 3C). Thus, CD44 expression cannot be used to
distinguish alloreactive cells from nonalloreactive memory cells in
this model.
We therefore next investigated whether the early activation marker CD69
could be used to identify alloreactive T cells. By 24 h after
adoptive transfer to an allogeneic recipient, the expression of CD69 on
donor T cells in bulk was elevated, and it remained elevated at 48
h before returning to baseline levels by 72 h (Fig. 3, A and B). By 48 h, 60% of CD4 T cells
were CD69 positive by division cycle 2, corresponding to maximal
expression, following which it began to diminish (Fig. 3, D
and E). These data indicate that even after extensive
reaction time, elevated CD69 expression distinguishes alloreactive
cells early in their proliferative history. However, in contrast to
CD44, the surface expression of CD69 by B6 Thy-1.1 donor T cells in
syngeneic hosts was low at all time points examined (Fig. 3A, insets). Therefore, although CD69 expression
varies with reaction time in bulk and with cell division at the single
cell level, its elevated expression in the allogeneic donor T cells,
and not the syngeneic controls, reliably distinguishes alloreactive T
cells independently of their proliferation.
Alloreactive T cell precursor frequency determination by CD69 expression
Based on the preceding observation that CD69 expression identifies
Ag-activated allogeneic T cells, we next investigated whether the
surface expression of CD69 48 h after adoptive transfer can be
used to quantify alloreactive T cells in vivo. As seen in Fig. 4, regions can be defined in a flow
cytometry plot indicating daughter cells resulting from precursors that
were activated and divided (Pdiv),
donor cells that were activated but did not divide
(Pact), and donor cells that did not
become activated and did not divide
(Pnil). Using this analysis,
44.60 ± 3.21% of total and 43.50 ± 2.44% of CD4 T cells
that were activated by allogeneic stimulation in vivo failed to
proliferate
(Pact/(Pact
+ Pnil)). Interestingly, these results
were consistent with our previous findings that roughly 40% of
adoptively transferred TCR-transgenic T cells failed to divide in an
immunized host (16, 17). Adjusting the results of the
alloreactive T cell frequency determined by method 3, the most accurate
method described, to account for this nonproliferative activated pool
yielded alloreactive T cell frequencies of roughly 12.4% and 10.1%
for total and CD4 T cells, respectively.
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Finally, we investigated whether nonalloreactive cells expressed
CD69 and/or proliferated in response to cytokines secreted by
alloreactive neighbors or by direct contact with them, so-called
bystander activation. CFSE-labeled syngeneic bystander
B6D2F1 splenocytes and lymph node cells were
coadoptively transferred with unlabeled C57BL/6 Thy-1.1 allogeneic
donor splenocytes and lymph node cells into a
B6D2F1 mouse. In control experiments,
CFSE-labeled bystander B6 splenocytes and lymph node cells were
coadoptively transferred with unlabeled C57BL/6 Thy-1.1 splenocytes and
lymph node cells into a B6 Thy-1.2 mouse. As expected, there was no
proliferation of donor cells in the autologous transfer (Fig. 5A), but considerable
activation of the Thy-1.1+ donors in the
allogeneic transfer (Fig. 5B). At 48 h, there was
negligible up-regulation of CD69 by the bystander cells in the
autologous adoptive transfer (Fig. 5C) and only 9.40 ±
4.67% of the bystanders expressed CD69 (Fig. 5D) in the
allogeneic transfer. The expression of CD69 by bystander T cells in the
allogeneic reaction was 10-fold less intense than the CD69
expression by the allogeneic donor cells. Importantly, in neither the
syngeneic nor the allogeneic transfer did the CFSE-labeled bystanders
proliferate (Fig. 5, E and F), despite induction
of CD69 expression in the latter instance.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The in vivo alloreactive T cell frequency was calculated by three different methods based on donor T cell recovery. In method 1, the precursor T cells that divided at 72 h are a fraction of the total donor precursor cells analyzed, yielding results that agree with studies examining splenocytes transferred to an irradiated allogeneic host (22), but are higher than most historical estimates of the alloreactive T cell frequency. The alloreactive response measured by method 1 most likely overrepresents proliferative donors because donor cells that do not find a niche, or do not divide, will have died by 72 h, and consequently are not analyzed. This analysis is useful for in vitro studies examining T cell responses to mitogen, in which all of the cells are potentially reactive, and all are recoverable at the time of analysis (16).
In method 2, we quantified alloreactivity at the opposite extreme, before donor cell proliferation occurred. The result is likely to be a low estimate of alloreactivity because recovery of donor cells from the recipient lymphoid organs is limited by cell death and by there being only a small number of transferred cells that may be circulating or residing in nonlymphoid organs at the time of analysis. Method 2 does, however, consider that the total potential donor T cell pool is larger than, and possibly biologically different from, its portion recovered from a recipient at 72 h, as done in method 1.
As a solution, method 3 expresses the maximal T cell proliferative response as a fraction of donor T cells that had engrafted in the recipient lymphoid organs, but had not begun to proliferate. By considering the extent of lymphoid engraftment, we believe the recovered precursor frequency measured by method 3 is the most biologically valid of the methods described. Indeed, the results obtained by this method are in close agreement with our in vitro MLR results, in which the alloreactive T cell precursor frequency is calculated based on a known number of potentially alloreactive T cells, all of which are recoverable at the time of analysis. The assumptions of this yield-independent calculation for the MLR are the same in method 3, in which the cells that proliferate in vivo are expressed as a fraction of the donor T cells that have homed to the lymphoid organs, where they are situated for alloantigen presentation.
We recognize a number of caveats to method 3. First, the observed alloresponse might not represent all donor T cells if alloreactive donor cells that engraft nonlymphoid organs behave differently from cells that populate the spleen and lymph nodes. By limiting analysis to the lymphoid organs, however, the maximal alloresponse is quantified. Second, the donor cells that do establish in the recipient lymphoid organs might not be representative of the total donor peripheral T cell population. However, we demonstrated with the alloantigen-specific bm12 model that the donor cells that home to the recipient secondary lymphoid tissues are quantitatively representative of all donor cells, and are not preferentially sequestered at these sites. Additionally, the established cells are similar in phenotype to the transferred cells, as the CD4 to CD8 ratio of the B6 donor cells recovered from B6D2F1 recipients at 24 h (2.10 ± 0.29) was comparable with that of injected donor cells (1.67 ± 0.12). Finally, donor cell death between 0 and 24 h might lead to a high estimate of the frequency of alloreactive T cells due to overrepresenting proliferative survivors. To address this, we are currently attempting to quantify and track the proliferative behavior of dead or dying adoptively transferred allogeneic T cells in vivo.
The in vivo alloreactive T cell precursor pool size can vary with the
model used for its measurement. Whether or not the host is irradiated
may affect the derived numbers. For example, in a
parentF1 model, such as was used in this
study, the intact host immune environment affords physiological
conditions for histocompatibility Ag presentation and costimulation of
donor lymphocytes by recipient APCs. Alternatively, in a model in which
the host is irradiated, the ablated immune compartment of the recipient
may be less likely to provide physiologic conditions for direct
alloantigen presentation or the delivery of costimulatory signals.
Moreover, any alloantigen-specific donor cell proliferation in an
irradiated host could be obscured by the nonspecific homeostatic
proliferation that occurs when a limited number of T cells are
transferred into immuno-incompetent mice (23, 24, 25). In the
model used in this study, there is no homeostatic proliferation, so the
measured pool size reflects only alloantigen-specific responses. The
irradiated recipient model may be appropriate for studies designed to
mimic the clinical situation of bone marrow transplantation, whereas
the parentF1 model may be better suited to
studies of clinical solid organ transplantation, in which the
alloimmune response occurs in the context of a normal immune
compartment.
The in vivo alloantigen-specific proliferation demonstrated marked heterogeneity in the number of rounds of division the precursor cells underwent by 72 h. How many times an alloreactive T cell divides may influence cell differentiation and effector function, as has been demonstrated in TCR-transgenic T cells responding to nominal Ag in vitro (26) and in vivo (17). This heterogeneity also suggests that the allogeneic response in vivo may consist of some T cells that proliferate, some that are anergic, and some that are deleted or die. The techniques described in this study would enable quantification of proliferating and nonproliferating cells under specific immunotherapeutic interventions to determine the relative contribution of these T cell responses. Furthermore, isolation and purification of these distinct subsets are possible for functional and biochemical characterization.
We found a substantial number of alloactivated cells did not divide by 48 h. This group, which comprised almost half of the donor cells that expressed CD69, would not be detected by any of the three quantitative methods described, as it is nonproliferative at the time of analysis. Interestingly, the fraction of alloactivated T cells that actually divided equaled 50–60%, a value similar to our previous findings quantifying the frequency of proliferating splenic T cells stimulated with mitogen or adoptively transferred TCR-transgenic T cells in an immunized host (16, 17). This suggests that even in the vigorous in vivo polyclonal response to alloantigen, individual T cell proliferation is stringently regulated by an intracellular checkpoint downstream from TCR ligation, and CD69 expression, that limits the total proliferative response.
This system allows the direct measurement of bystander activation, a problematic factor in previous attempts to determine the alloreactive precursor frequency. The supernatant of a primary MLR was shown to induce the proliferation, measured by 3[H]thymidine incorporation, of naive cells equivalent to the primary response itself (15). In contrast, bystander phenomena has been shown to lead to activation-induced cell death, in nonalloreactive donors (27). In our in vivo system, by 48 h no syngeneic transferred bystanders had proliferated, and only 9.40 ± 4.67% expressed the early activation marker CD69, while the alloreactive donor cells had up-regulated CD69 to a greater extent and demonstrated considerable proliferation. These data suggest that bystander activity is negligible in allogeneic proliferative responses occurring in vivo.
Accurately quantifying allogeneic responses is important to investigations of peripheral tolerance induction to alloantigen. In a haplotype mismatch cardiac allograft model, recipients treated with agents to block costimulatory signals and rapamycin alone or in combination led to a reduction in alloreactive T cell clonal size through apoptosis. However, cyclosporin A in combination with costimulatory blockade abolished T cell proliferation, apoptosis, and tolerance induction (28, 29). Future clinical strategies to induce transplant tolerance may therefore rely on quantifying the alloreactive T cell pool size that may be eliminated as well as the pool size of alloreactive cells that may be immunoregulatory and therefore lead to enduring tolerance in the absence of ongoing pharmacological intervention.
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Acknowledgments |
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Footnotes |
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2 A.D.W. and L.A.T. are co-senior authors.
3 Address correspondence and reprint requests to Dr. Laurence A. Turka, 700 Clinical Research Building, 415 Curie Boulevard, Philadelphia, PA 19104.
4 Abbreviations used in this paper: LDA, limiting dilution analysis; ELISPOT, enzyme-linked immunospot.
Received for publication June 19, 2000. Accepted for publication October 25, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cell receptor. Science 281:835.This article has been cited by other articles:
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  P. Kumar, A. Vahedi-Faridi, W. Saenger, E. Merino, J. A. Lopez de Castro, B. Uchanska-Ziegler, and A. Ziegler Structural Basis for T Cell Alloreactivity among Three HLA-B14 and HLA-B27 Antigens J. Biol. Chem., October 23, 2009; 284(43): 29784 - 29797. [Abstract] [Full Text] [PDF]
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 L. M. Moldenhauer, K. R. Diener, D. M. Thring, M. P. Brown, J. D. Hayball, and S. A. Robertson Cross-Presentation of Male Seminal Fluid Antigens Elicits T Cell Activation to Initiate the Female Immune Response to Pregnancy J. Immunol., June 15, 2009; 182(12): 8080 - 8093. [Abstract] [Full Text] [PDF]
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B. H. Koehn, M. L. Ford, I. R. Ferrer, K. Borom, S. Gangappa, A. D. Kirk, and C. P. Larsen PD-1-Dependent Mechanisms Maintain Peripheral Tolerance of Donor-Reactive CD8+ T Cells to Transplanted Tissue J. Immunol., October 15, 2008; 181(8): 5313 - 5322. [Abstract] [Full Text] [PDF]
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E. S. Yolcu, X. Gu, C. Lacelle, H. Zhao, L. Bandura-Morgan, N. Askenasy, and H. Shirwan Induction of Tolerance to Cardiac Allografts Using Donor Splenocytes Engineered to Display on Their Surface an Exogenous Fas Ligand Protein J. Immunol., July 15, 2008; 181(2): 931 - 939. [Abstract] [Full Text] [PDF]
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T. Fehr, F. Haspot, J. Mollov, M. Chittenden, T. Hogan, and M. Sykes Alloreactive CD8 T Cell Tolerance Requires Recipient B Cells, Dendritic Cells, and MHC Class II J. Immunol., July 1, 2008; 181(1): 165 - 173. [Abstract] [Full Text] [PDF]
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S. Manicassamy, D. Yin, Z. Zhang, L. L. Molinero, M.-L. Alegre, and Z. Sun A Critical Role for Protein Kinase C-{theta}-Mediated T Cell Survival in Cardiac Allograft Rejection J. Immunol., July 1, 2008; 181(1): 513 - 520. [Abstract] [Full Text] [PDF]
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M. L. Ford, M. E. Wagener, S. S. Hanna, T. C. Pearson, A. D. Kirk, and C. P. Larsen A Critical Precursor Frequency of Donor-Reactive CD4+ T Cell Help Is Required for CD8+ T Cell-Mediated CD28/CD154-Independent Rejection J. Immunol., June 1, 2008; 180(11): 7203 - 7211. [Abstract] [Full Text] [PDF]
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M. Hu, D. Watson, G. Y. Zhang, N. Graf, Y. M. Wang, M. Sartor, B. Howden, J. Fletcher, and S. I. Alexander Long-Term Cardiac Allograft Survival across an MHC Mismatch after "Pruning" of Alloreactive CD4 T Cells J. Immunol., May 15, 2008; 180(10): 6593 - 6603. [Abstract] [Full Text] [PDF]
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A. Kroemer, X. Xiao, M. D. Vu, W. Gao, K. Minamimura, M. Chen, T. Maki, and X. C. Li OX40 Controls Functionally Different T Cell Subsets and Their Resistance to Depletion Therapy J. Immunol., October 15, 2007; 179(8): 5584 - 5591. [Abstract] [Full Text] [PDF]
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 R. Girlanda and A. D. Kirk Frontiers in Nephrology: Immune Tolerance to Allografts in Humans J. Am. Soc. Nephrol., August 1, 2007; 18(8): 2242 - 2251. [Abstract] [Full Text] [PDF]
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M. AbuAttieh, M. Rebrovich, P. J. Wettstein, Z. Vuk-Pavlovic, A. H. Limper, J. L. Platt, and M. Cascalho Fitness of Cell-Mediated Immunity Independent of Repertoire Diversity J. Immunol., March 1, 2007; 178(5): 2950 - 2960. [Abstract] [Full Text] [PDF]
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 M. L. Ford, B. H. Koehn, M. E. Wagener, W. Jiang, S. Gangappa, T. C. Pearson, and C. P. Larsen Antigen-specific precursor frequency impacts T cell proliferation, differentiation, and requirement for costimulation J. Exp. Med., February 19, 2007; 204(2): 299 - 309. [Abstract] [Full Text] [PDF]
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 M. A. Brehm, J. Mangada, T. G. Markees, T. Pearson, K. A. Daniels, T. B. Thornley, R. M. Welsh, A. A. Rossini, and D. L. Greiner Rapid quantification of naive alloreactive T cells by TNF-{alpha} production and correlation with allograft rejection in mice Blood, January 15, 2007; 109(2): 819 - 826. [Abstract] [Full Text] [PDF]
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E. A. Rowell, L. Wang, W. W. Hancock, and A. D. Wells The Cyclin-Dependent Kinase Inhibitor p27kip1 Is Required for Transplantation Tolerance Induced by Costimulatory Blockade J. Immunol., October 15, 2006; 177(8): 5169 - 5176. [Abstract] [Full Text] [PDF]
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N. Najafian, M. J. Albin, and K. A. Newell How Can We Measure Immunologic Tolerance in Humans? J. Am. Soc. Nephrol., October 1, 2006; 17(10): 2652 - 2663. [Abstract] [Full Text] [PDF]
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S. Laffont, J. D. Coudert, L. Garidou, L. Delpy, A. Wiedemann, C. Demur, C. Coureau, and J.-C. Guery CD8+ T-cell-mediated killing of donor dendritic cells prevents alloreactive T helper type-2 responses in vivo Blood, October 1, 2006; 108(7): 2257 - 2264. [Abstract] [Full Text] [PDF]
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 M. L. Drakes, S. J. Czinn, and T. G. Blanchard Regulation of Murine Dendritic Cell Immune Responses by Helicobacter felis Antigen. Infect. Immun., August 1, 2006; 74(8): 4624 - 4633. [Abstract] [Full Text] [PDF]
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E. Landais, A. Morice, H. M. Long, T. A. Haigh, B. Charreau, M. Bonneville, G. S. Taylor, and E. Houssaint EBV-Specific CD4+ T Cell Clones Exhibit Vigorous Allogeneic Responses J. Immunol., August 1, 2006; 177(3): 1427 - 1433. [Abstract] [Full Text] [PDF]
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 L.A. Bergmeier and T. Lehner Innate and Adaptive Mucosal Immunity in Protection against HIV Infection Advances in Dental Research, April 1, 2006; 19(1): 21 - 28. [Abstract] [Full Text] [PDF]
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J. S. Obhrai, M. H. Oberbarnscheidt, T. W. Hand, L. Diggs, G. Chalasani, and F. G. Lakkis Effector T Cell Differentiation and Memory T Cell Maintenance Outside Secondary Lymphoid Organs J. Immunol., April 1, 2006; 176(7): 4051 - 4058. [Abstract] [Full Text] [PDF]
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B. Koehn, S. Gangappa, J. D. Miller, R. Ahmed, and C. P. Larsen Patients, pathogens, and protective immunity: the relevance of virus-induced alloreactivity in transplantation. J. Immunol., March 1, 2006; 176(5): 2691 - 2696. [Abstract] [Full Text] [PDF]
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N. J. Felix, A. Suri, J. J. Walters, S. Horvath, M. L. Gross, and P. M. Allen I-Ep-Bound Self-Peptides: Identification, Characterization, and Role in Alloreactivity J. Immunol., January 15, 2006; 176(2): 1062 - 1071. [Abstract] [Full Text] [PDF]
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M. Mesel-Lemoine, M. Cherai, S. Le Gouvello, M. Guillot, V. Leclercq, D. Klatzmann, V. Thomas-Vaslin, and F. M. Lemoine Initial depletion of regulatory T cells: the missing solution to preserve the immune functions of T lymphocytes designed for cell therapy Blood, January 1, 2006; 107(1): 381 - 388. [Abstract] [Full Text] [PDF]
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 M. L. Drakes, T. G. Blanchard, and S. J. Czinn Colon lamina propria dendritic cells induce a proinflammatory cytokine response in lamina propria T cells in the SCID mouse model of colitis J. Leukoc. Biol., December 1, 2005; 78(6): 1291 - 1300. [Abstract] [Full Text] [PDF]
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L. Wang, R. Han, I. Lee, A. S. Hancock, G. Xiong, M. D. Gunn, and W. W. Hancock Permanent Survival of Fully MHC-Mismatched Islet Allografts by Targeting a Single Chemokine Receptor Pathway J. Immunol., November 15, 2005; 175(10): 6311 - 6318. [Abstract] [Full Text] [PDF]
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 L. A. Bergmeier, K. Babaahmady, Y. Wang, and T. Lehner Mucosal alloimmunization elicits T-cell proliferation, CC chemokines, CCR5 antibodies and inhibition of simian immunodeficiency virus infectivity J. Gen. Virol., August 1, 2005; 86(8): 2231 - 2238. [Abstract] [Full Text] [PDF]
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A. M. E. Whitelegg, L. E. M. Oosten, S. Jordan, M. Kester, A. G. S. van Halteren, J. A. Madrigal, E. Goulmy, and L. D. Barber Investigation of Peptide Involvement in T Cell Allorecognition Using Recombinant HLA Class I Multimers J. Immunol., August 1, 2005; 175(3): 1706 - 1714. [Abstract] [Full Text] [PDF]
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L. M. Reed-Loisel, B. A. Sullivan, O. Laur, and P. E. Jensen An MHC Class Ib-Restricted TCR That Cross-Reacts with an MHC Class Ia Molecule J. Immunol., June 15, 2005; 174(12): 7746 - 7752. [Abstract] [Full Text] [PDF]
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M. A. Brehm, K. A. Daniels, J. R. Ortaldo, and R. M. Welsh Rapid Conversion of Effector Mechanisms from NK to T Cells during Virus-Induced Lysis of Allogeneic Implants In Vivo J. Immunol., June 1, 2005; 174(11): 6663 - 6671. [Abstract] [Full Text] [PDF]
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S. E. Sandner, M. R. Clarkson, A. D. Salama, A. Sanchez-Fueyo, C. Domenig, A. Habicht, N. Najafian, H. Yagita, M. Azuma, L. A. Turka, et al. Role of the Programmed Death-1 Pathway in Regulation of Alloimmune Responses In Vivo J. Immunol., March 15, 2005; 174(6): 3408 - 3415. [Abstract] [Full Text] [PDF]
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 T. Suzuki, T. Fukuhara, M. Tanaka, A. Nakamura, K. Akiyama, T. Sakakibara, D. Koinuma, T. Kikuchi, R. Tazawa, M. Maemondo, et al. Vaccination of Dendritic Cells Loaded with Interleukin-12-Secreting Cancer Cells Augments In vivo Antitumor Immunity: Characteristics of Syngeneic and Allogeneic Antigen-Presenting Cell Cancer Hybrid Cells Clin. Cancer Res., January 1, 2005; 11(1): 58 - 66. [Abstract] [Full Text] [PDF]
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D. Watson, G. Y. Zhang, M. Sartor, and S. I. Alexander "Pruning" of Alloreactive CD4+ T Cells Using 5- (and 6-)Carboxyfluorescein Diacetate Succinimidyl Ester Prolongs Skin Allograft Survival J. Immunol., December 1, 2004; 173(11): 6574 - 6582. [Abstract] [Full Text] [PDF]
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D. Kreisel, A. M. Krasinskas, A. S. Krupnick, A. E. Gelman, K. R. Balsara, S. H. Popma, M. Riha, A. M. Rosengard, L. A. Turka, and B. R. Rosengard Vascular Endothelium Does Not Activate CD4+ Direct Allorecognition in Graft Rejection J. Immunol., September 1, 2004; 173(5): 3027 - 3034. [Abstract] [Full Text] [PDF]
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M. Drakes, T. Blanchard, and S. Czinn Bacterial Probiotic Modulation of Dendritic Cells Infect. Immun., June 1, 2004; 72(6): 3299 - 3309. [Abstract] [Full Text] [PDF]
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 M. Y. Mapara and M. Sykes Tolerance and Cancer: Mechanisms of Tumor Evasion and Strategies for Breaking Tolerance J. Clin. Oncol., March 15, 2004; 22(6): 1136 - 1151. [Abstract] [Full Text] [PDF]
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D. M. Richards, S. L. Dalheimer, B. D. Ehst, T. L. Vanasek, M. K. Jenkins, M. I. Hertz, and D. L. Mueller Indirect Minor Histocompatibility Antigen Presentation by Allograft Recipient Cells in the Draining Lymph Node Leads to the Activation and Clonal Expansion of CD4+ T Cells That Cause Obliterative Airways Disease J. Immunol., March 15, 2004; 172(6): 3469 - 3479. [Abstract] [Full Text] [PDF]
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W. R. Godfrey, M. R. Krampf, P. A. Taylor, and B. R. Blazar Ex vivo depletion of alloreactive cells based on CFSE dye dilution, activation antigen selection, and dendritic cell stimulation Blood, February 1, 2004; 103(3): 1158 - 1165. [Abstract] [Full Text] [PDF]
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I. Lee, L. Wang, A. D. Wells, Q. Ye, R. Han, M. E. Dorf, W. A. Kuziel, B. J. Rollins, L. Chen, and W. W. Hancock Blocking the Monocyte Chemoattractant Protein-1/CCR2 Chemokine Pathway Induces Permanent Survival of Islet Allografts through a Programmed Death-1 Ligand-1-Dependent Mechanism J. Immunol., December 15, 2003; 171(12): 6929 - 6935. [Abstract] [Full Text] [PDF]
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T. J. Lang, P. Nguyen, J. C. Papadimitriou, and C. S. Via Increased Severity of Murine Lupus in Female Mice Is Due to Enhanced Expansion of Pathogenic T Cells J. Immunol., December 1, 2003; 171(11): 5795 - 5801. [Abstract] [Full Text] [PDF]
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A. R. Rao, M. P. Quinones, E. Garavito, Y. Kalkonde, F. Jimenez, C. Gibbons, J. Perez, P. Melby, W. Kuziel, R. L. Reddick, et al. CC Chemokine Receptor 2 Expression in Donor Cells Serves an Essential Role in Graft-versus-Host-Disease J. Immunol., November 1, 2003; 171(9): 4875 - 4885. [Abstract] [Full Text] [PDF]
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M. A. Brehm, T. G. Markees, K. A. Daniels, D. L. Greiner, A. A. Rossini, and R. M. Welsh Direct Visualization of Cross-Reactive Effector and Memory Allo-Specific CD8 T Cells Generated in Response to Viral Infections J. Immunol., April 15, 2003; 170(8): 4077 - 4086. [Abstract] [Full Text] [PDF]
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A. E. Troy and H. Shen Cutting Edge: Homeostatic Proliferation of Peripheral T Lymphocytes Is Regulated by Clonal Competition J. Immunol., January 15, 2003; 170(2): 672 - 676. [Abstract] [Full Text] [PDF]
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S. Rutella, L. Pierelli, G. Bonanno, S. Sica, F. Ameglio, E. Capoluongo, A. Mariotti, G. Scambia, G. d'Onofrio, and G. Leone Role for granulocyte colony-stimulating factor in the generation of human T regulatory type 1 cells Blood, September 18, 2002; 100(7): 2562 - 2571. [Abstract] [Full Text] [PDF]
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A. G. S. Buggins, G. J. Mufti, J. Salisbury, J. Codd, N. Westwood, M. Arno, K. Fishlock, A. Pagliuca, and S. Devereux Peripheral blood but not tissue dendritic cells express CD52 and are depleted by treatment with alemtuzumab Blood, August 13, 2002; 100(5): 1715 - 1720. [Abstract] [Full Text] [PDF]
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E. Chiffoleau, G. Beriou, P. Dutartre, C. Usal, J.-P. Soulillou, and M. C. Cuturi Role for Thymic and Splenic Regulatory CD4+ T Cells Induced by Donor Dendritic Cells in Allograft Tolerance by LF15-0195 Treatment J. Immunol., May 15, 2002; 168(10): 5058 - 5069. [Abstract] [Full Text] [PDF]
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F. Haspot, F. Villemain, G. Laflamme, F. Coulon, D. Olive, J. Tiollier, J.-P. Soulillou, and B. Vanhove Differential effect of CD28 versus B7 blockade on direct pathway of allorecognition and self-restricted responses Blood, March 15, 2002; 99(6): 2228 - 2234. [Abstract] [Full Text] [PDF]
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A. Sanchez-Fueyo, M. Weber, C. Domenig, T. B. Strom, and X. X. Zheng Tracking the Immunoregulatory Mechanisms Active During Allograft Tolerance J. Immunol., March 1, 2002; 168(5): 2274 - 2281. [Abstract] [Full Text] [PDF]
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S. Maury, B. Salomon, D. Klatzmann, and J. L. Cohen Division rate and phenotypic differences discriminate alloreactive and nonalloreactive T cells transferred in lethally irradiated mice Blood, November 15, 2001; 98(10): 3156 - 3158. [Abstract] [Full Text] [PDF]
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F. Sebille, K. Gagne, M. Guillet, N. Degauque, A. Pallier, S. Brouard, B. Vanhove, M.-A. Delsuc, and J.-P. Soulillou Direct Recognition of Foreign MHC Determinants by Naive T Cells Mobilizes Specific V{beta} Families Without Skewing of the Complementarity-Determining Region 3 Length Distribution J. Immunol., September 15, 2001; 167(6): 3082 - 3088. [Abstract] [Full Text] [PDF]
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S. J. Bensinger, A. Bandeira, M. S. Jordan, A. J. Caton, and T. M. Laufer Major Histocompatibility Complex Class II-Positive Cortical Epithelium Mediates the Selection of Cd4+25+ Immunoregulatory T Cells J. Exp. Med., August 20, 2001; 194(4): 427 - 438. [Abstract] [Full Text] [PDF]
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D. A. Mandelbrot23, K. Kishimoto, H. Auchincloss Jr., A. H. Sharpe, and M. H. Sayegh Rejection of Mouse Cardiac Allografts by Costimulation in trans J. Immunol., August 1, 2001; 167(3): 1174 - 1178. [Abstract] [Full Text] [PDF]
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P. Marrack, J. Bender, M. Jordan, W. Rees, J. Robertson, B. C. Schaefer, and J. Kappler Presidential Address to The American Association of Immunologists : Major Histocompatibility Complex Proteins and TCRs: Do They Really Go Together Like a Horse and Carriage? J. Immunol., July 15, 2001; 167(2): 617 - 621. [Full Text] [PDF]
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) and CD44 (
) expressed as relative mean fluorescence
intensity (MFI) in arbitrary units and the percentage of cells positive
for CD69 (
) and CD44 (
) are shown. The results are representative
of three separate experiments.
