Phenotypic CD8+ T Cell Diversification
Occurs before, during, and after the First T
Cell Division
This information is current as
of June 15, 2017.
Fabrice Lemaître, Hélène D. Moreau, Laura Vedele and
Philippe Bousso
J Immunol published online 8 July 2013
http://www.jimmunol.org/content/early/2013/07/08/jimmun
ol.1300424
http://www.jimmunol.org/content/suppl/2013/07/08/jimmunol.130042
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Supplementary
Material
Published July 8, 2013, doi:10.4049/jimmunol.1300424
The Journal of Immunology
Phenotypic CD8+ T Cell Diversification Occurs before,
during, and after the First T Cell Division
Fabrice Lemaı̂tre,*,† Hélène D. Moreau,*,†,‡ Laura Vedele,*,† and Philippe Bousso*,†
D4+ and CD8+ T cell effector responses are generated
from the progenies of individual Ag-specific T cells that
proliferate and differentiate upon Ag recognition. Effector T cell pools are typically highly heterogeneous as revealed
by multiparametric flow cytometry or single-cell PCR, and comprise cells with variable phenotypes and functional properties (1–
5). The recent development of cytometry by time-of-flight (6) has
provided an even more dramatic illustration of this complexity,
revealing the existence of .200 distinct memory CD8+ T cell
phenotypes (7). Effector and memory T cell diversity may promote the efficacy and flexibility of immune responses when dealing
with a wide variety of infectious agents (8).
T cell heterogeneity could result from two complementary processes. First, interclonal diversification reflects the possibility
that distinct Ag-specific T cell clones adopt different fates early on
(e.g., prior to the first cell division) that will be imprinted in their
progenies. By imaging the activity of a fluorescent IFN-g reporter
in T cells in vivo, we recently noted extensive heterogeneity in
reporter activity before the first cell division (9). Second, intraclonal diversification permits generation of varied T cell phenotypes from a single T cell precursor. A striking example was
provided by a study that followed the fate of single adoptively
transferred CD8+ T cells (10) and revealed the generation of both
effector and central memory T cells. Similar conclusions were
reached in a study that used an elegant barcoding system to track
the fate of individual T cell clones (11). TCR repertoire analyses
also suggested that the progeny of a single T cell could contribute
C
*Dynamics of Immune Responses Unit, Institut Pasteur, 75015 Paris, France;
†
INSERM U668, 75015 Paris, France; and ‡University Paris Diderot, Sorbonne Paris
Cité, Cellule Pasteur, F-75015 Paris, France
Received for publication February 12, 2013. Accepted for publication June 9, 2013.
This work was supported by Institut Pasteur, INSERM, Fondation pour la Recherche
Médicale, and a European Research Council starting grant (LymphocyteContacts).
Address correspondence and reprint requests to Dr. Philippe Bousso, Dynamics of
Immune Responses Unit, Institut Pasteur, 25 Rue du Dr Roux, 75015 Paris, France.
E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: CFP, cyan fluorescent protein; DC, dendritic cell;
WT, wild-type.
Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300424
to phenotypically distinct T cell subsets (12, 13). Stochastic events
occurring after the first T cell division, such as Ag re-encounter
(14) or exposure to a distinct cytokine milieu, may account for
intraclonal diversification. In addition, it has recently been proposed that the first T cell division occurs in an asymmetric fashion
(15–17), a process that could involve asymmetric proteasome segregation (18) and have the potential to generate phenotypic and
functional diversity within a T cell progeny.
Although evidence for both intraclonal as well as interclonal
diversification exists, there is little information regarding their respective contribution during T cell activation. In addition, the
possible contribution of divergent fate after the first T cell division
remains to be precisely quantified. To address these issues, we used
a short-term cloning strategy that allowed us to analyze the phenotypic and functional diversity in the progeny of single T cells
activated by dendritic cells (DCs). To further assess whether the
first T cell division contributes to intraclonal T cell diversity, we
isolated paired daughter cells and compared the composition of their
progenies. Finally, we developed an adoptive T cell transfer strategy
under limiting conditions to track the fate of individual T cell
progenies in vivo. Our results support the idea that T cell diversification occurs before, during, and after the initial T cell division.
Materials and Methods
Mice
C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA). Rag2/2 OT-I and GFP- or cyan fluorescent protein (CFP)–
expressing Rag2/2 OT-I TCR transgenic mice were bred in our animal
facility. All mice were housed in our animal facility under specific pathogenfree conditions. Animal experiments were performed in accordance with
institutional guidelines for animal care and use.
Cell purification
CD44lowCD8+ T cells were purified from lymph nodes and spleens of
Rag2/2 OT-I mice by negative selection using the CD8+ T cell isolation
kit (Miltenyi Biotec, Paris, France) and a biotinylated anti-mouse CD44
mAb (clone IM7; eBioscience, San Diego, CA). DCs were prepared from
spleens of wild-type (WT) C57BL/6 mice [either untreated or injected i.p.
with 100 mg poly(I:C) 5 h before] by positive selection using CD11c+
microbeads (Miltenyi Biotec) after collagenase digestion. Unless stated
otherwise, DCs were pulsed with 1028 M OVA257–264 peptide (SIINFEKL;
Polypeptide Group, Strasbourg, France) for 10 min at room temperature
and washed extensively. When indicated, the lower-affinity Q4 OVA variant peptide was used (SIIQFEKL).
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Effector T cell responses rely on a phenotypically and functionally heterogeneous population of cells. Whether this diversity is
programmed before clonal expansion or in later phases as a result of stochastic events or asymmetric cell division is not fully understood. In this study, we first took advantage of a sensitive in vitro assay to analyze the composition of single CD8+ T cell progenies.
Heterogeneity was predominantly observed between progenies of distinct clones, but could also be detected within individual
progenies. Furthermore, by physically isolating daughter cells of the first T cell division, we showed that differences in paired
daughter cell progenies contributed to intraclonal diversification. Finally, we developed an in vivo limiting dilution assay to
compare individual T cell progenies following immunization. We provided evidence for simultaneous intraclonal and interclonal
diversification in vivo. Our results support the idea that T cell diversification is a continuous process, initiated before clonal
expansion and amplified during the first and subsequent cell divisions. The Journal of Immunology, 2013, 191: 000–000.
2
MECHANISMS OF T CELL DIVERSIFICATION
Short-term progeny analysis
Purified naive OT-I cells (2 3 10 cells) were activated with OVA-pulsed
DCs (2 3 106 cells) for 24 h at 37˚C in 24-well culture plates. Activated
OT-I cells were then cloned by limiting dilution at 0.5 cell/well into
6
60-well minitray culture plates (Nunc, Rochester, NY), in 10 ml complete
RPMI 1640 media containing 25 U/ml human rIL-2 (Roche, Meylan,
France). After cloning, each well was examined under the microscope, and
only wells containing a single T cell after cloning were further analyzed.
The absence of DCs after T cell cloning was systematically confirmed by
FIGURE 2. T cell phenotypic heterogeneity originates from both interclonal and intraclonal diversification. (A) CD25 and CD62L expression on representative individual T cell progenies. (B) Graphs compiling levels of CD25 and CD62L in individual progenies. Each column corresponds to one progeny. Each
dot corresponds to one cell. Cells pooled from all progenies are shown in green. In (C), values obtained in (B) were randomized and assigned to virtual progenies.
(D) Individual progenies have a more limited diversity than the bulk T cell population. Each red dot shows the ratio between the SD for CD25 (or CD62L)
expression of one progeny and the SD of the pooled population. Values ,100% indicate lower overall diversity. Similar values were calculated with virtual
progenies in 10 independent randomizations (black dots). (E) A fraction of T cell progenies displays high intraclonal phenotypic diversity. The normalized SD
for CD25 expression in each progeny is graphed against the normalized SD for CD62L. Blue and black dots correspond to heterogenous and homogenous
progenies, respectively. Thresholds were defined using virtual progenies, which all displayed diverse profiles. Representative of six independent experiments.
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FIGURE 1. Analyzing the short-term progeny of individual T cells by flow cytometry. (A) Experimental setup. Naive OT-I
T cells were stimulated with peptide-pulsed
DCs for 24 h and cloned in 10 ml wells.
After additional 3 d of culture, cells recovered from individual wells were analyzed by flow cytometry. (B) Representative
images showing an individual well immediately after cloning and following 3 d of
culture. (C–E) A 1:1 mixture of WT and
CFP+ naive OT-I T cells was stimulated and
cloned at 0.5 cell/well (C). Wells containing
a single cell (D) or multiple cells (E) were
analyzed by flow cytometry. (F) Recovery
of T cells after flow cytometry. The number of cells in individual T cell progenies
counted under the microscope is graphed
against the number of live lymphocytes detected by flow cytometry. (G) Growth curves
of individual progenies were obtained by
counting the content of each well under the
microscope every day. Representative of
three independent experiments.
The Journal of Immunology
microscopic examination. Culture plates were incubated for 3 d at 37˚C.
On day 4, progenies containing .30 cells were analyzed by flow cytometry. In some experiments, minitray culture plates were coated at room
temperature for 5 h with PBS containing 10 mg/ml recombinant Kb-OVA
(19) and purified anti-CD28 mAb (eBioscience; clone 37.51). Purified
naive OT-I CD8+ T cells were directly cloned at 0.5 cell/well into those
coated minitray culture plates, in presence of rIL-2, and cultured for
4 d before FACS analysis. For the analysis of daughter cell progenies,
wells that contained a single T cell at the time of cloning were examined
12–14 h later for the occurrence of their first cell division. Paired daughter
cells were physically separated by splitting the well content in two distinct
wells, and ensuring that each well contained one of the daughter cells
(otherwise the process was repeated). Daughter cell progenies were cultured for additional 3 d and analyzed by flow cytometry.
Adoptive T cell transfer under limiting conditions
Naive CD8+ T cells were purified from Rag2/2 OT-I (CD45.2) and Rag2/2
OT-I GFP+ (CD45.2) mice and mixed at a 1:1 ratio. The indicated number
of T cells was adoptively transferred into CD45.1 recipients that were
injected in the footpad the next day with 2 3 106 DCs pulsed with 1 3 1027
M OVA257–264 peptide. Four days later, the draining and nondraining lymph
nodes were harvested. The totality of lymph node cells was stained and
analyzed by flow cytometry.
3
Pont de Claix, France). Allophycocyanin-conjugated anti-human granzyme B (clone GB11) was purchased from Invitrogen (Carlsbad, CA).
T cell progenies were transferred into FACS tube containing 50 ml diluted
mAb and incubated at 4˚C for 20 min, before direct analysis on a FACSCanto II cytometer (BD Biosciences). Intracellular cytokine stainings were
performed using the Cytofix/Cytoperm kit (BD Biosciences) following
stimulation with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of brefeldin A (1 mg/ml). Given the small size of our samples, we
controlled for the absence of cell contamination during FACS acquisition
by including samples containing FACS buffer only.
Data processing and statistical analyses
Flow cytometry
R-PE–conjugated anti-CD25 (clone PC61), allophycocyanin-conjugated or
allophycocyanin-eFluor 780-conjugated anti-CD62L (clone Mel-14), and
PE-Cy7–conjugated anti-CD8 (clone 53-6.7) were purchased from eBioscience. Brilliant violet 421–conjugated anti-CD45.2 was purchased from
BioLegend (San Diego, CA). PerCP-Cy5.5–conjugated anti-CD25 (clone
PC61), PE-conjugated anti–IFN-g (clone XMG1.2), and PE-conjugated
anti-Vb5.1/5.2 (clone MR9-4) were purchased from BD Biosciences (Le
Results
Analyzing single T cell progenies by flow cytometry
To dissect the origin of T cell diversity, we used a short-term in vitro
assay aimed to analyze single T cell progenies (9). In this system,
naive OT-I T cells are stimulated with peptide-pulsed DCs for 24 h
and then cloned at 0.5 cell/well in 10 ml microwells (Fig. 1A).
FIGURE 3. T cell functional heterogeneity originates from both interclonal and intraclonal diversification. (A) Short-term T cell progenies were subjected to
intracellular staining for granzyme B and IFN-g following restimulation with PMA/ionomycin. FACS profile showing IFN-g and granzyme B staining in three
individual progenies. (B and C) Interclonal variability in IFN-g and granzyme B intracellular content. Graphs compiling levels of IFN-g and granzyme B in
individual progenies (B). Each column corresponds to one progeny. Each dot corresponds to a single cell. Cells pooled from all progenies are shown in green. In
(C), values obtained in (B) were randomized and assigned to virtual progenies. (D) Individual progenies have a more limited diversity than the bulk T cell
population. Each red dot shows the ratio between the SD for IFN-g or granzyme B levels of one progeny and the SD of the pooled population. Values ,100%
indicate lower overall diversity. In parallel, similar values were calculated with virtual progenies in 10 independent randomizations (black dots). (E) A fraction
of T cell progenies displays high intraclonal phenotypic diversity. For each progeny, the normalized SD for IFN-g staining expression in the progeny is graphed
against the normalized SD for granzyme B. Blue and black dots correspond to heterogenous and homogenous progenies, respectively. Thresholds were defined
using virtual progenies, which all displayed diverse profiles. Results are representative of three independent experiments.
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For each progeny, the percentage of intraclonal variation was determined
by dividing its SD by the SD of pooled progenies. Statistical significance
was assessed by comparing these values with a target value of 100% using
a one-sample t test (Prism version 5.0; GraphPad Software, La Jolla, CA).
We created virtual progenies (whose size matched that of the experimental data) by randomizing fluorescence values from all progenies of an entire experiment using the “randbetween” function from Excel software
(Microscoft, Issy-Les-Moulineaux, France). To compare paired daughter
cell progenies, mean fluorescence intensity of each progeny was compared
with an unpaired t test using Prism software. Normalized SDs were obtained by dividing the SD for one parameter by the corresponding geometric mean fluorescence.
4
After examination under the microscope, only wells containing a
single T cell were further analyzed. After additional 3 d of culture,
the early progenies of a single T cell would typically comprise 30–
200 cells that could be analyzed by flow cytometry (Fig. 1A, 1B).
To ensure cells recovered in individual wells originated from a
single T cell, we cloned a mixture of WT and CFP-expressing
OT-I T cells (Fig. 1C). Progenies recovered from wells that initially contained a single T cell were uniformly CFP+ or CFP2, confirming their clonal origin (Fig. 1D). This was not the case for
wells in which two or more cells were initially detected, as these
wells often contained a mixture of CFP+ and CFP2 cells, as expected (Fig. 1E). These results confirmed that our approach offers
the ability to analyze single T cell progenies following a single
round of activation, in the absence of restimulation or prolonged
culture period. We observed a good correlation between the T cell
numbers counted under the microscope and the number of events
recorded by flow cytometry (Fig. 1F), with a typical recovery
efficacy of 50%. Interestingly, substantial diversity was detected at
the level of clone size and growth curves (Fig. 1G).
To analyze the phenotypic diversity within and between clonal
T cell progenies, we analyzed the expression of the following two
important markers: CD25, which plays a crucial role in effector
T cell differentiation (20, 21), and CD62L, which regulates T cell
homing properties (22, 23). When individual progenies were
compared, we observed considerable clone-to-clone variations
(Fig. 2A, 2B). Of note, levels of CD25 and CD62L staining
appeared independent of the total progeny size (Supplemental Fig.
1). To exclude the possibility that these differences were attributable to the small size of the cell population analyzed, we randomized the values of CD25 (and CD62L) expression compiled
FIGURE 4. Differences in the progenies of paired
daughter T cells contribute to intraclonal T cell diversity. (A) Experimental setup. Naive OT-I T cells
were stimulated with peptide-pulsed DCs for 24 h and
cloned in 10 ml microwells. Wells containing a single
T cell at the time of cloning were examined again after
12–14 h for the occurrence of the first cell division.
Paired daughter cells were transferred into two separate wells and cultured for 72 h, and their progenies
were analyzed by FACS. (B) Overlaid FACS profiles
showing the distribution of CD25 and CD62L markers
for paired daughter T cells. (C) The distribution of
CD25 and CD62L expression was compared between
paired daughter T cell progenies. The percentage of
significantly different paired progenies is indicated for
each marker. Data are compiled from four experiments
and correspond to 41 paired progenies. Values measured in paired daughter cell progenies were randomized and assigned to two virtual paired progenies.
Randomization abrogated differences between paired
progenies. (D) Five examples of paired progenies
found to be either significantly different or similar.
Progenies from paired daughter cells are shown in red
and blue. Each dot corresponds to one cell. Corresponding randomized paired progenies are shown in
black for comparison. Representative of four independent experiments.
from all progenies to generate virtual progenies. Randomized
progenies no longer exhibited clone-to-clone variation (Fig. 2C).
The variability in experimental progenies appeared significantly
(p , 0.05) lower on average than the total diversity (all clones
combined), but this was not the case for virtual progenies in 10
independent randomizations (Fig. 2D). These results confirmed
that the observed interclonal diversity for CD25 and CD62L
molecules is not the result of sampling limitations. In sum, our
results support the idea that heterogeneity in CD25 and CD62L
expression on T cells originates from the addition of clonal
progenies that tend to be less diverse than the bulk of the population and differs from one another. These results support the
view that phenotypic diversification is initiated before the first cell
division.
Another striking observation was that the overall diversity for
each of the markers largely varied from clone to clone. As shown
in Fig. 2E, some T cell clones displayed homogenous expression
of both markers, whereas others were more diverse for one or both
markers. Thus, some T cell clones (80 of 135, 59%) displayed
extensive phenotypic diversification as they expanded.
Similar results were obtained when the analysis of T cell progenies was performed at days 3 and 6, indicating that T cell diversity
is detected at multiple time points (Supplemental Fig. 2). Altogether, our results support the idea that interclonal variations as
well as intraclonal diversity in a subset of T cell clones contribute
to the phenotypic heterogeneity of effector T cells.
Phenotypic T cell diversification arises in a variety of
stimulation conditions
To test whether the large T cell heterogeneity observed was a peculiarity of our experimental system, we repeated the experiments
using DCs pulsed with different peptide doses or with the Q4 OVA
variant peptide that displayed a lower affinity for the OT-I TCR. As
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Measuring interclonal and intraclonal phenotypic diversity
in T cells
MECHANISMS OF T CELL DIVERSIFICATION
The Journal of Immunology
shown in Supplemental Fig. 3, pronounced phenotypic diversification was observed in these conditions. We also assessed the role
of DC maturation by analyzing the progenies of T cells stimulated
by activated DCs that were isolated from poly(I:C)-treated mice.
Again, a high level of intraclonal and interclonal diversity was seen
in this setting (Supplemental Fig. 4A). Likewise, phenotypic diversification was evident when single naive T cells were cloned
directly into microwells coated with recombinant pMHC and antiCD28 (Supplemental Fig. 4B). Thus, both interclonal and intraclonal diversification appear to be a general hallmark of T cell
activation, largely independent of the stimulation conditions used.
Interclonal and intraclonal diversity in T cell production of
IFN-g and granzyme B content
FIGURE 5. Intraclonal and interclonal phenotypic
diversification of CD8+ T cells both operate in vivo.
(A) Experimental setup. Decreasing number of naive
CD45.2 GFP+ and GFP2 OT-I T cells (1:1 mixture)
was transferred into CD45.1 recipients that were injected in the footpad with OVA-pulsed DCs. Four days
later, the totality of cells from the draining lymph node
was analyzed by flow cytometry. OT-I T cells were
identified as CD45.2+ CD8+ Vb5+. (B) Immunization
with DCs results in expansion and phenotypic diversification of GFP+ and GFP2 OT-I T cells. FACS plots
show CD25 and CD62L expression on gated OT-I
T cells 4 d after transfer of 1 3 105 naive OT-I T cells
and immunization with unpulsed or OVA-pulsed DCs.
(C) Adoptive T cell transfer under limiting conditions.
Mice were transferred with the indicated number of
cells from GFP+:GFP2 OT-I T cell mixture and immunized with peptide-pulsed DCs. Pie charts depict
the probability that an expanded OT-I GFP+ or GFP2 T
cell population could be detected in the draining lymph
node in various conditions of T cell transfer. (D)
Number of OT-I T cells detected 4 d postimmunization
in nondraining and draining lymph nodes after transfer
of 1 3 103 OT-I T cells. (E) Percentage of GFP+ cells
among OT-I T cells (CD45.2+) detected in draining
lymph nodes. (F) Representative FACS plots showing
the composition of the expanded OT-I T cell population for the indicated condition of T cell transfer.
(G) Representative FACS profiles showing CD25 and
CD62L expression on GFP+ OT-I T cells in limiting
conditions of adoptive transfer (1 3 103 OT-I T cells).
(H) Overlaid FACS profiles of GFP2 and GFP+ OT-I
T cells detected in the same lymph node in limiting
conditions of adoptive transfer (1 3 103 OT-I T cells).
One example of divergent (left) and one example of
similar (right) pattern are shown. (I) The distribution of
CD25 expression was compared between GFP2 and
GFP+ T cell populations shown in (H). Representative
of four independent experiments. *p , 0.05, **p ,
0.01, ***p , 0.001.
Granzyme B content also varied from clone to clone. Variations
in IFN-g and granzyme B were significant, as demonstrated by
the lack of differences in randomized progenies (Fig. 3B, 3C).
Again, the diversity in IFN-g and granzyme B levels in an individual progeny was, on average, significantly lower than that
of the total population (Fig. 3D). Of note, a subset of clones (23
of 39, 59%) showed diversified expression of at least one of the
molecules examined (Fig. 3E).
Distinct contribution of paired daughter T cells can promote
intraclonal diversity
We next assessed the origin of intraclonal T cell diversity observed
in some of the progenies. Specifically, we asked whether the first
cell division contributed to this diversification by comparing the
fate of paired daughter cells. Subcloning of daughter or granddaughter cells has been used previously to compare the fate of
a given T cell clone cultured under distinct cytokine milieu (24,
25). Thus, we thought to physically separate daughter T cells
immediately following the first cell division and monitor their
respective fate using our short-term progeny assay (Fig. 4A). For
approximately half of the T cell clones, CD25 and CD62L were
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Next, we extended our analysis to functional T cell markers using
intracellular staining. We found that only a fraction of the T cells
within a given progeny produced IFN-g in response to restimulation (Fig. 3A). Moreover, the frequency of IFN-g–producing
cells and the level of IFN-g production varied between distinct
progenies (Fig. 3A, 3B). Thus, heterogeneity in IFN-g production
was the result of both interclonal and intraclonal variations.
5
6
expressed at similar levels in the progeny of each daughter cell.
For the remaining clones, paired daughter cells generated progenies that significantly differed in their levels of CD25 and/or
CD62L (Fig. 4B–D). Virtual daughter cell progenies (generated by
data randomization) were not significantly different from one
another, confirming that the differences detected are not the result
of sampling limitations (Fig. 4C, 4D). Our results indicate that the
first cell division most likely contributes to intraclonal diversity in
a fraction of T cell clones.
Monitoring intraclonal and interclonal T cell diversity in vivo
FIGURE 6. A model for the generation of T cell
heterogeneity during clonal expansion. (A–C) Previously proposed models for the diversification of T
cells. (A) T cell fate is determined early on (prior to
clonal expansion) and transmitted to the progeny. (B)
T cells adopt distinct fates late during the expansion
phase. (C) Asymmetric cell division during first division generates two possible fates within a progeny. (D)
Continuous diversification generates a heterogeneous
pool of T cells. Our data support a model in which T
cell diversification is initiated prior to cell division and
amplified during the first and subsequent divisions.
with an average of 71 6 10 OT-I T cells being recovered when
totality of the draining lymph node was analyzed by flow cytometry
(Fig. 5D). No T cell expansion could be detected when 1 3 102
cells were transferred (Fig. 5C). Whereas the ratio of GFP+:GFP2
OT-I T cells was close to 1 in immunized mice transferred with 1 3
105 and 1 3 104 T cells, it was highly variable upon transfer of 1 3
103 cells (Fig. 5E, 5F), most likely reflecting the limited number of
T cell clones recruited. Thus, very few (typically 0, 1, or 2) GFP+
and GFP2 T cells are present in the popliteal lymph node upon
transfer of 1 3 103 cells, allowing us to detect the progenies of one
or very few cells in the GFP+ and GFP2 subsets after immunization.
Under limiting conditions, expanded T cells could still harbor a
diverse profile of CD25 and CD62L expression, suggesting intraclonal diversification (Fig. 5G). In addition, we also noted variations
between the T cell populations recovered from different mice, and,
most importantly, we could also observe differences when the
phenotype of expanded T cells in the GFP+ and GFP2 populations
was compared in the same lymph node (Fig. 5H, 5I). These differences were significant, as they were abolished by randomization of pooled values. Thus, distinct T cell clones could generate
different progenies of effectors in the same microenvironment,
providing evidence for interclonal diversification in vivo.
Discussion
In the present work, we mapped the origin of phenotypic diversification during the course of T cell activation. Using a sensitive
in vitro assay, we identified three checkpoints in the generation of
a heterogeneous pool of effector T cells. First, large interclonal
variability revealed that events occurring prior to the first cell
division have a strong impact on the fate of T cell progenies. What
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To test the in vivo relevance of some of our findings, we developed
an in vivo assay in which the fate of a very limited number of T cells
could be tracked in immunized mice. To validate this approach, we
first transferred a large cohort (1 3 105 cells) of GFP+ and GFP2
OT-I T cells (both expressing CD45.2 and mixed at a 1:1 ratio) in
CD45.1 recipients. Mice were then injected s.c. with OVA-pulsed
DCs (Fig. 5A). On day 4, GFP+ and GFP2 had similarly expanded
in the draining lymph node (and had not yet recirculated), and GFP+
and GFP2 effector T cells displayed a similarly diverse pattern
of CD25 and CD62L expression (Fig. 5B). We reasoned that, by
progressively decreasing the number of transferred T cells, we
should reach a stage at which only one or very few GFP+ and
GFP2 T cells would be present and stimulated in the draining
lymph node. Whereas we could systematically detect T cell expansion in both GFP+ and GFP2 negative subsets when 1 3 105
and 1 3 104 T cells were transferred, this was no longer the case
with the transfer of 1 3 103 T cells, suggesting that we had reached
limiting conditions (Fig. 5C). In these conditions, GFP+ and GFP2
T cell expansion was detected in 72% of the lymph nodes analyzed,
MECHANISMS OF T CELL DIVERSIFICATION
The Journal of Immunology
Acknowledgments
We thank members of the Bousso laboratory for comments on the manuscript; H. Beuneu for the generation of OT-I CFP mice; and C. Auriau,
N. Yatim, and M.L. Albert for providing mice.
Disclosures
The authors have no financial conflicts of interest.
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could be the origin of T cell interclonal diversification? These
differences may originate from variation in TCR and cytokine
signals (26) experienced by each T cell clone. Alternatively, differences could pre-exist in naive T cells. In this respect, it has
been shown that even monoclonal T cells exhibit cell-to-cell
variation in CD8 and soluble hematopoietic phosphatase 1 levels
and that these differences accounted for the diversity in response
to TCR stimulation (27). Second, we found that approximately
half of the T cell clones analyzed exhibited significant diversity
in phenotypic and functional markers, indicating that late events
encompassing the successive divisions contribute to effector T cell
heterogeneity. Although challenging, it will be of importance to
clarify why some T cell progenies end up more homogenous than
others. Third, through physical separation of paired daughter
T cells, we provided evidence that T cell diversification can be
initiated in the context of the first T cell division. Differences in
paired daughter cells may originate from an asymmetric cell division, as recently proposed (15–18), or alternatively from random
events occurring just after the cell division, such as variability in
exposure to cytokines. We focused our analysis on two important
markers, CD25 and CD62L, whose expression levels regulate T
cell functional responses. The differences in CD25 expression
observed in our experiments (up to 100-fold) are meaningful and
expected to have substantial functional consequences, as predicted
by previous studies (20). Of note, T cell phenotypic diversification
appears to be a general phenomenon, observed with different
peptide doses, affinities, or stimulating conditions. To study the
composition of T cell progenies in vivo, we have also introduced
an in vivo limiting dilution assay combined with the use of differently labeled T cell populations. In this approach, we decreased
the number of transferred T cells to the point where, in most cases,
a single T cell is activated in the popliteal lymph node draining the
site of immunization. This methodology revealed that one T cell
can give rise to a phenotypically diverse population of effectors
(intraclonal diversification) in vivo, a finding consistent with a
previous report in which single T cells were micromanipulated
for adoptive transfer. Our approach is unique in that it allowed us
to compare, in the same lymph node, two subsets (GFP2 and
GFP+) originating from a limited number of clones. Divergent
phenotypic profiles could be observed between GFP+ and GFP2
subsets under limiting conditions (but not when large number of
T cells was transferred), an observation that implies a significant
level of interclonal differences in vivo and is consistent with two
very recent studies that used barcoding and lineage tracing in vivo
(28, 29). Thus, intraclonal and interclonal diversifications operate
simultaneously in vivo.
Several models have been proposed to explain T cell heterogeneity, including early commitment, late T cell diversification,
and asymmetric cell division (Fig. 6A–C). In the present study, our
ability to analyze the composition of numerous T cell progenies
and assess the fate of daughter cells enabled us to estimate simultaneously the contribution of these different mechanisms.
Altogether, our results suggest that functional T cell diversification is a continuous process (Fig. 6D) largely initiated prior to
clonal expansion, but later amplified during the first and subsequent cell divisions. Lastly, the in vitro and in vivo methodologies
described in this work should provide new opportunities to track
and dissect the development of a variety of T cell subsets during
clonal expansion.
7
8
multipotentiality through many cell divisions. Proc. Natl. Acad. Sci. USA 94:
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effector site contains cells with flexible cytokine profiles. J. Exp. Med. 190:
1081–1092.
26. Feinerman, O., G. Jentsch, K. E. Tkach, J. W. Coward, M. M. Hathorn,
M. W. Sneddon, T. Emonet, K. A. Smith, and G. Altan-Bonnet. 2010. Single-cell
quantification of IL-2 response by effector and regulatory T cells reveals critical
plasticity in immune response. Mol. Syst. Biol. 6: 437.
MECHANISMS OF T CELL DIVERSIFICATION
27. Feinerman, O., J. Veiga, J. R. Dorfman, R. N. Germain, and G. Altan-Bonnet.
2008. Variability and robustness in T cell activation from regulated heterogeneity
in protein levels. Science 321: 1081–1084.
28. Gerlach, C., J. C. Rohr, L. Perié, N. van Rooij, J. W. van Heijst, A. Velds,
J. Urbanus, S. H. Naik, H. Jacobs, J. B. Beltman, et al. 2013. Heterogeneous
differentiation patterns of individual CD8+ T cells. Science 340: 635–639.
29. Buchholz, V. R., M. Flossdorf, I. Hensel, L. Kretschmer, B. Weissbrich,
P. Gräf, A. Verschoor, M. Schiemann, T. Höfer, and D. H. Busch. 2013.
Disparate individual fates compose robust CD8+ T cell immunity. Science
340: 630–635.
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