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Clonal Competition Inhibits the Proliferation
and Differentiation of Adoptively Transferred
TCR Transgenic CD4 T Cells in Response to
Infection
Kathryn E. Foulds and Hao Shen
J Immunol 2006; 176:3037-3043; ;
doi: 10.4049/jimmunol.176.5.3037
http://www.jimmunol.org/content/176/5/3037
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References
The Journal of Immunology
Clonal Competition Inhibits the Proliferation and
Differentiation of Adoptively Transferred TCR Transgenic
CD4 T Cells in Response to Infection1
Kathryn E. Foulds2 and Hao Shen3
D
uring the course of a primary T cell response, rare precursor T cells proliferate and expand in an Ag-specific
manner to acquire effector function and generate memory cells. Mounting evidence suggests that the mechanisms that
regulate these processes are different for CD4 and CD8 T cells. For
example, CD8 T cells are capable of dividing, differentiating, and
becoming memory cells without further antigenic stimulation after
an initial activation with Ag (1–3). However, while continued
stimulation with Ag is not required during CD4 T cell expansion
(4 – 6), repeated Ag encounters are necessary for the differentiation
and optimal survival of CD4 T cells (4). CD4 and CD8 T cells
seem to expand and contract synchronously in response to infection; however, CD8 T cell memory appears to be stable, while
CD4 T cell memory gradually declines (7). In addition, recent
reports suggest that CD4 T cells that secrete IFN-␥ are short-lived
and do not develop into memory cells (8), while memory CD8 T
cells arise from IFN-␥-producing effectors (9).
The initial response of Ag-specific T cells is difficult to analyze
because the precursor frequency of specific cells is too low to
visualize by conventional methods. Thus, most studies have examined the primary responses of CD4 and CD8 T cells in vitro by
stimulation with anti-CD3 mAb or in vivo by adoptive transfer of
CFSE-labeled TCR transgenic cells (10). These studies have
shown that the CD4 T cell response is heterogeneous in nature
with responding cells dividing and differentiating to various extents (11–19), while the CD8 T cell response is more homogeneous
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104
Received for publication May 13, 2004. Accepted for publication December 23, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grant AI-45025 (to H.S.).
2
Current address: Cellular Immunology Section, Vaccine Research Center, National
Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,
MD 20892.
3
Address correspondence and reprint requests to Dr. Hao Shen, Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104.
E-mail address: [email protected]
Copyright © 2006 by The American Association of Immunologists, Inc.
with all responding cells dividing and differentiating extensively (1–
3). The reason for these differences between CD4 and CD8 T cells is
not known, and it remains to be seen whether these differences also
occur during the response to an infection in normal hosts. In vitro
stimulation with mAbs to CD3 and CD28 in the presence of IL-2 may
not effectively simulate the complex inflammatory environment that
occurs during infection (20). For example, important cytokines and
costimulatory molecules that are available in vivo may not be provided appropriately in in vitro settings (21). This is particularly important to consider when comparing the responses of CD4 and CD8
T cells because CD4 T cells have been shown to be more dependent
on costimulation than CD8 T cells (22–35). Similarly, it is not known
whether the response of a large number of adoptively transferred
monoclonal TCR transgenic T cells accurately reflects the response of
Ag-specific T cells at a physiological precursor frequency. Thus, it
remains to be determined whether the reported differences in CD4 and
CD8 T cell division and differentiation reflect the true nature of CD4
and CD8 T cell responses.
To investigate the differences in CD4 and CD8 T cell proliferation and differentiation in response to a natural infection, we developed an adoptive transfer system that enabled us to study polyclonal CD4 and CD8 T cells in vivo during the course of a primary
Listeria monocytogenes (LM)4 infection. Surprisingly, we observed that adoptively transferred polyclonal CD4 and CD8 T cells
proliferated extensively in response to LM infection. These findings contrast strikingly with previous results that TCR transgenic
CD4 T cells divide a limited number of times in response to infection, while TCR transgenic CD8 T cells divide extensively under the same conditions. We hypothesized that the extent of monoclonal TCR transgenic CD4 T cell division was limited due to
clonal competition between CD4 T cells of the same specificity
imposed by an abnormally high precursor frequency. Indeed, we
found that reducing the number of adoptively transferred TCR
transgenic CD4 T cells increased the number of divisions completed by these cells and the percentage of differentiated effectors
produced by these cells. As we approached physiological precursor
frequencies, TCR transgenic CD4 T cells divided as extensively as
4
Abbreviations used in this paper: LM, Listeria monocytogenes; LLO, listeriolysin O.
0022-1767/06/$02.00
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CD4 and CD8 T cells have been shown to proliferate and differentiate to different extents following antigenic stimulation. CD4 T
cells form a heterogenous pool of effector cells in various stages of division and differentiation, while nearly all responding CD8 T
cells divide and differentiate to the same extent. We examined CD4 and CD8 T cell responses during bacterial infection by adoptive
transfer of CFSE-labeled monoclonal and polyclonal T cells. Monoclonal and polyclonal CD8 T cells both divided extensively, whereas
monoclonal CD4 T cells underwent limited division in comparison with polyclonal CD4 T cells. Titration studies revealed that the limited
proliferation of transferred monoclonal CD4 T cells was due to inhibition by a high precursor frequency of clonal T cells. This unusually
high precursor frequency of clonal CD4 T cells also inhibited the differentiation of these cells. These results suggest that the adoptive
transfer of TCR transgenic CD4 T cells significantly underestimates the extent of proliferation and differentiation of CD4 T cells
following infection. The Journal of Immunology, 2006, 176: 3037–3043.
3038
INHIBITION OF CD4 T CELL RESPONSE BY CLONAL COMPETITION
polyclonal CD4 T cells. In addition, the protection provided by
these cells increased as the number of transferred cells decreased.
Our results suggest that the adoptive transfer of TCR transgenic
CD4 T cells may significantly underestimate the extent of proliferation and differentiation of CD4 T cells in response to infection.
Materials and Methods
Mice and bacterial strains
OT-I, OT-II, and BALB/c-TgN(DO11.10)Loh mice were previously described (36 –38). OT-I and OT-II mice were bred onto the B6.PL-Thy-1.1
background. C57BL/6 and B6.Ly-5.2/Cr (Ly-5.1) mice were obtained from
the National Cancer Institute (Bethesda, MD). B6.PL-Thy-1.1 mice were
obtained from The Jackson Laboratory, and BALB/c-Thy-1.1 mice were
obtained from C. Surh (The Scripps Institute, La Jolla, CA). Construction
of rLM-OVA was described previously (11, 39). The LD50 of rLM-OVA
in BALB/c mice is ⬃5 ⫻ 104, and the LD50 of rLM-OVA in C57BL/6 mice
is ⬃5 ⫻ 105; experimental mice were infected with 0.1 LD50 of rLM-OVA.
Adoptive transfer
Flow cytometry
Proliferation of transferred cells was visualized by incremental loss of
CFSE fluorescence. Transferred OT-I and OT-II cells were identified by
staining with mAb to Thy-1.1, V␣2, and CD8 or CD4, respectively.
DO11.0 cells were identified by staining with mAbs to Thy-1.2, CD4, and
KJ1-26. For the OT-II and DO11.10 titration experiment, 3-fold more
events were acquired for each dilution, starting with 1 million events for
4.5 million transferred cells. For the unlabeled competitors experiment, the
same number of events was acquired for each dilution of competitors (10
Bacterial loads
Three titrations of splenocytes from OT-II mice containing 1.5 ⫻ 106,
1.5 ⫻ 105, and 1.5 ⫻ 104 OVA-specific CD4 T cells were adoptively
transferred into C57BL/6 mice. One day later, the recipients were infected
with 1 ⫻ 105 CFU rLM-OVA, i.v. Bacterial loads were determined 3 days
later by plating 10-fold serial dilutions of spleen and liver homogenates in
1% Triton X-100/PBS on brain heart infusion agar plates (Difco). Bacterial
numbers were log transformed before statistical analyses were conducted
by t tests (two tailed, 95% confidence intervals) using GraphPad Prism
(GraphPad).
Results
Adoptively transferred polyclonal CD4 T cells divide extensively
in comparison with monoclonal CD4 T cells
To compare the proliferation of CD4 and CD8 T cells in the context of a natural infection, we developed an adoptive transfer system to visualize primary polyclonal T cell responses. CFSE-labeled CD4 and CD8 T cells from C57BL/6 mice were adoptively
transferred into Thy-1.2 or Ly-5.2 congenic mice. For comparison,
we also adoptively cotransferred CFSE-labeled monoclonal TCR
transgenic CD4 and CD8 T cells from OT-II and OT-I mice (2 ⫻
106 specific cells each), respectively. Recipient mice were subsequently infected with rLM expressing OVA (rLM-OVA). As seen
before, OT-II T cells divided to a limited extent with a spectrum of
responding cells in each division, while OT-I T cells divided extensively with most of the responding cells becoming CSFE negative, indicating seven or more divisions (Fig. 1A). However,
FIGURE 1. Adoptively transferred polyclonal CD4 T cells undergo extensive division in comparison with monoclonal CD4 T cells. CFSE-labeled OT-II
and OT-I cells were cotransferred (2 ⫻ 106 each) into congenic B6.Thy-1.2 mice (A), and CFSE-labeled splenocytes (2 ⫻ 107) from B6.Ly-5.1 mice were
transferred into congenic B6.Ly-5.2 recipients (B). One day later, the recipients were infected with rLM-OVA, and the response of donor cells was analyzed
on days 3 or 5, and 8 postinfection. The cells from the uninfected recipients were analyzed 9 days posttransfer. A, Proliferation of transferred OT-II
(Thy-1.1⫹/V␣2⫹/CD4⫹) and OT-I (gated on Thy-1.1⫹/V␣2⫹/CD8⫹) cells as visualized by loss of CFSE. B, Proliferation of polyclonal donor CD4
(CD4⫹/Ly-5.1⫹/Ly-5.2⫺) and CD8 (CD8⫹/Ly-5.1⫹/Ly-5.2⫺) T cells. C, Absolute numbers of CSFE-negative polyclonal donor CD4 and CD8 T cells per
spleen on days 5 and 8. D, CD44 expression on CFSE-negative (black line) and CFSE-positive (shaded plots) polyclonal donor CD4 and CD8 T cells on
day 8 postinfection. E, Percentages of host CD4 and CD8 T cells with an activated (CD44high) phenotype on days 0, 5, and 8. Plots in A, B, and D are from
a representative mouse, and data in C and E are the mean and SD of each group (five mice per group). Similar results were also obtained with the adoptive
transfer of polyclonal T cells using Thy-1.1 donor and Thy-1.2 recipient mice.
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Splenocytes from OT-I, OT-II, DO11.10, or Ly-5.1 mice were labeled with
CFSE, as described (40). For OT-I/OT-II combined transfers, T cells were
enriched by depleting splenocytes with B220 and MHC class II MicroBeads by MACS (Miltenyi Biotec) and CFSE labeled, and the indicated
number of each cell type was cotransferred per mouse. For the Ly-5.1 and
individual OT-II and DO11.10 transfers, total splenocytes were labeled
with CFSE and the indicated number of splenocytes was transferred. Experimental groups included two to five mice per group, and similar results
were observed in at least two independent experiments.
million events) because the number of CFSE-labeled cells remained the
same for each dilution. Ly-5.1 cells were identified by staining with mAbs
to Ly-5.1 and CD4 or CD8; cells that stained positive for Ly-5.2 were
excluded. Three million events/mouse were acquired to visualize responding cells. All mAbs were obtained from BD Pharmingen, except KJ1-26,
which was obtained from Caltag Laboratories. Intracellular IFN-␥ staining
was performed with the Cytofix/Cytoperm Plus kit (BD Pharmingen) after
5 h of in vitro stimulation with 1 ␮M OVA257–264, 3 ␮M OVA323–339,
or 3 ␮M listeriolysin O (LLO)190 –201 peptides.
The Journal of Immunology
Adoptively transferred polyclonal T cells become activated and
differentiate to the same extent as host T cells
We reasoned that the response of adoptively transferred polyclonal
T cells was more likely to mimic the endogenous response of host
T cells than the response of adoptively transferred monoclonal T
cells. However, it is still possible that adoptively transferred T
cells may become activated during ex vivo manipulation or may
not engraft properly. We therefore examined whether the response
of the adoptively transferred polyclonal T cells accurately reflected
the response of the endogenous host T cells by comparing the
activation and differentiation of the donor and host polyclonal T
cells. Donor CD4 and CD8 T cells were activated to the same
extent as recipient CD4 and CD8 T cells in naive mice and on days
5 and 8 postinfection, as measured by increased surface expression
FIGURE 2. The response of adoptively transferred polyclonal T cells
closely mimics that of endogenous host T cells. Splenocytes from B6.Ly5.1 mice were transferred into B6.Ly-5.2 mice, and the response of donor
and host T cells was analyzed on days 5 and 8 after rLM-OVA infection.
A, Activation of donor and host CD4 and CD8 T cells was assessed by
staining for CD44. The shaded and open histograms represent donor (Ly5.1⫹) and host (Ly-5.2⫹) cells, respectively, and the numbers indicate the
percentage of donor/host CD4 or CD8 T cells that are CD44high. B, Agspecific responses by donor and host CD4 and CD8 T cells were measured
by intracellular IFN-␥ staining. Shown are the percentages of donor or host
CD4 T cells that are LLO190 specific and CD8 T cells that are OVA257
specific. Data represent the mean and the SD of five mice per group.
of CD44 (CD44high) (Fig. 2A). For example, at the peak of the
response on day 8 postinfection, 73.4 and 70.0% of donor and host
CD4 T cells were activated, respectively, and 72.1 and 70.4% of
donor and host CD8 T cells were activated, respectively. No statistical differences occurred between donor and host T cells at any
time point examined. These results demonstrate that the adoptively
transferred T cells followed the same activation kinetics as endogenous T cells.
We also examined the Ag-specific responses of donor and recipient CD4 and CD8 T cells by intracellular IFN-␥ staining on
days 5 and 8 postinfection (Fig. 2B). CD4 T cells mount a broad
response to many different LM epitopes; however, we examined
the response to LLO190 –201 because it is clearly immunodominant
(41). The donor and host CD4 T cell responses to LLO190 –201 were
nearly the same for both time points. Approximately 2% of both
donor and host CD4 T cells were LLO190 specific on day 5. This
increased to 9% at the peak of the response on day 8. Likewise,
both the donor and host CD8 T cell responses to OVA257–264 were
almost identical. Approximately 1% of both donor and host CD8
T cells were OVA257 specific on day 5, increasing to ⬃4% on day
8. No statistical differences were observed between donor and recipient T cells for either T cell subset on the two time points
examined. Thus, the same levels of Ag-specific CD4 and CD8 T
cells were generated from the donor and host T cell populations.
The activation and Ag-specific response data show that adoptively transferred polyclonal T cells behave like the endogenous T
cells in response to LM infection. Thus, CD4 T cells responding to
infection most likely undergo extensive proliferation, as seen with
adoptively transferred polyclonal CD4 T cells, rather than limited
proliferation, as seen with TCR transgenic CD4 T cells.
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much to our surprise, polyclonal CD4 T cells divided to the same
extent as polyclonal CD8 T cells with most of the responding cells
dividing seven or more times (Fig. 1B). Thus, we observed a difference in the extent of division between adoptively transferred
monoclonal CD4 T cells and polyclonal CD4 T cells. Monoclonal
CD4 T cells divided a limited number of times in response to
infection, while all responding polyclonal CD4 T cells divided
extensively. In contrast, both monoclonal and polyclonal CD8 T
cells divided extensively in response to infection with the vast
majority of responding CD8 T cells becoming CFSE negative (Fig.
1, A and B).
Although the division profiles of polyclonal CD4 and CD8 T
cells are similar at the peak of the response on day 8, CFSEnegative CD8 T cells accumulated more rapidly than CFSE-negative CD4 T cells (Fig. 1B). On day 5 postinfection, 1.6 ⫻ 105
CFSE-negative CD4 T cells were present compared with 4.1 ⫻ 105
CFSE-negative CD8 T cells (Fig. 1C). However, at the peak of the
response on day 8, the total numbers of CFSE-negative CD4 and
CD8 T cells were nearly the same, 7.4 ⫻ 105 and 6.2 ⫻ 105 cells,
respectively. These results indicate that the CD8 T cell response is
greater than the CD4 T cell response early on, yet the magnitude
of total CD4 and CD8 T cell responses reaches comparable levels
at the peak of the response.
The CFSE-negative polyclonal CD4 and CD8 T cells had increased expression of the activation marker CD44 (Fig. 1D),
CD25, and CD132 (data not shown) in comparison with the nonresponding CFSE-positive cells, consistent with them having responded to antigenic stimulation. We also compared the activation
of host CD4 and CD8 T cells over the course of infection (Fig. 1E).
Naive mice contained nearly equal percentages of activated/memory (CD44high) CD4 and CD8 T cells, 30 and 34%, respectively.
By day 5 postinfection, the CD8 T cell subset had more activated
cells (⬃65% CD44high) than the CD4 T cell subset (⬃45%
CD44high). However, by the peak of the response on day 8, both
CD4 and CD8 T cells contained ⬃70% activated cells. These results are consistent with the above CFSE data showing that more
donor CD8 T cells had accumulated by day 5, yet similar numbers
of CFSE-negative CD4 and CD8 T cells were present by day 8
(Fig. 1C). Together, these results strongly indicate that, while more
activated CD8 T cells are present early during the course of the
immune response, the total CD4 T cell response is as robust as the
CD8 T response.
In summary, the adoptive transfer of CFSE-labeled splenocytes
enabled the simultaneous visualization of polyclonal CD4 and
CD8 T cell responses during a primary LM infection. Our results
show that polyclonal CD4 T cells, like CD8 T cells, divide extensively in response to LM infection. These results also reveal a
discrepancy between the proliferative responses of adoptively
transferred monoclonal and polyclonal CD4 T cells.
3039
3040
INHIBITION OF CD4 T CELL RESPONSE BY CLONAL COMPETITION
FIGURE 3. The extent of proliferation by adoptively transferred monoclonal CD4 T cells is inversely
related to the initial precursor frequency. A, The indicated number of CFSE-labeled OT-II or DO11.10 cells
was transferred into recipient mice, which were then
infected with rLM-OVA. Proliferation of donor cells
was visualized by loss of CFSE on days 3 and 8 postinfection for OT-II cells and on day 8 for DO11.10 cells.
B, CFSE-labeled OT-II and OT-I cells (2 ⫻ 105 each,
Thy-1.1⫹) were transferred, together with the indicated
amounts of unlabeled (competitor) Thy-1.2⫹ OT-II and
OT-I cells, into congenic Thy-1.2 mice. Proliferation of
CSFE-labeled OT-I (Thy-1.1⫹V␣2⫹CD8⫹) and OT-II
(Thy-1.1⫹V␣2⫹CD4⫹) cells was visualized on day 8
postinfection with rLM-OVA.
We considered the possibility that the limited division of the
monoclonal CD4 T cells was due to clonal competition imposed by
a high precursor frequency of cells with the same specificity.
Therefore, we transferred 3-fold serial dilutions of OT-II TCR
transgenic CD4 T cells into recipient mice and examined the extent
of division of these cells in response to infection with rLM-OVA.
Graded levels of transferred cells were present in the spleens of
uninfected control mice, as expected. In mice infected with rLMOVA, we found that the transfer of decreasing amounts of TCR
transgenic CD4 T cells resulted in more extensive division of these
cells (Fig. 3A). As seen before, the OT-II cells were nearly equally
distributed in divisions 1 through 7 in mice that received the largest number of transferred cells (4.5 ⫻ 106 OVA-specific CD4 T
cells) on day 8 postinfection. This trend was apparent as early as
day 3 postinfection. In contrast, the majority of OT-II cells responding to infection in the mice that received the lowest number
of transferred cells (1.5 ⫻ 105 OVA-specific CD4 T cells) were
CFSE negative on day 8 postinfection, resembling the proliferative
pattern of the polyclonal CD4 T cells seen in Fig. 1B. Similar
results were also seen with DO11.10 cells, which are on the
BALB/c background and are specific for the same CD4 T cell
epitope of OVA as OT-II cells (Fig. 3). We have shown previously
that the DO11.10 cells that proliferate in response to rLM-OVA infection do so in an Ag-specific manner because there is no proliferation of the TCR transgenic cells in response to infection with wildtype LM (11). Furthermore, use of the KJ1-26 clonotypic Ab in this
model ensures that the CFSE-negative cells are indeed Ag specific.
The above experimental design required the FACS acquisition
of increasingly more cells as the number of transferred cells decreased to have sufficient donor cells for analysis. To exclude the
potential biases this may introduce, we next cotransferred CFSElabeled OT-II and OT-I TCR transgenic T cells (2 ⫻ 105 specific
cells each) with increasing numbers of an unlabeled mixture of
purified OT-II and OT-I splenocytes, and then infected the recipients with rLM-OVA. This allowed us to follow the proliferation
of the same number of labeled TCR transgenic CD4 and CD8 T
cells in the face of increasing numbers of clonal T cells in the same
mouse. TCR transgenic CD4 T cells underwent fewer rounds of
division with increasing numbers of unlabeled OT-II competitors
(Fig. 3B), as seen when large numbers of labeled OT-II cells were
transferred (Fig. 3A). In contrast, OT-I TCR transgenic CD8 T
cells underwent at least seven rounds of division even in the presence of high numbers of unlabeled OT-I competitors (Fig. 3B). A
direct comparison between CD4 and CD8 T cells is difficult to
make because the number of class I and class II OVA epitopes
presented during infection with rLM-OVA is not likely the same,
and the affinity of the OT-II TCR is weaker than the OT-I TCR.
Nevertheless, these results demonstrate that the extent of proliferation of CD4 T cells is greatly inhibited by high precursor frequencies and suggest that the differences between the proliferation
of adoptively transferred monoclonal and polyclonal CD4 T cells
are due to the different precursor frequencies of Ag-specific clonal
T cells in the two systems. In addition, these results indicate that
the presence of CD4 T cells in various stages of division (Fig. 1A)
is the result of an abnormally high precursor frequency and may
not occur under physiological conditions.
High precursor frequencies of TCR transgenic CD4 T cells do
not rapidly clear bacteria from the spleens and livers of mice
The limited proliferation of transferred monoclonal CD4 T cells
could also be the result of less antigenic stimulation due to the
rapid clearance of bacteria by the large number of Ag-specific
FIGURE 4. The adoptive transfer of increasing numbers of TCR transgenic CD4 T cells does not result in decreasing numbers of bacteria. The
indicated number of CFSE-labeled OT-II cells was transferred into recipient mice, which were then infected with 1 ⫻ 105 CFU rLM-OVA. Bacterial loads were obtained from spleens and livers of mice 3 days postinfection. The line represents the mean of three mice/group. ⴱ, p ⬍ 0.05.
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Reducing the precursor frequency of monoclonal CD4 T cells
results in a proliferative profile similar to polyclonal CD4 T cells
The Journal of Immunology
clonal T cells present. Therefore, we examined the level of rLMOVA recovered from the spleens and livers of mice receiving
1.5 ⫻ 106, 1.5 ⫻ 105, and 1.5 ⫻ 104 OVA-specific OT-II TCR
transgenic CD4 T cells on day 3 postinfection. Similar numbers of
bacteria were present in the spleens of mice receiving 1.5 ⫻ 106,
1.5 ⫻ 105, and 1.5 ⫻ 104 OT-II T cells, while bacterial loads were
decreased by ⬃5-fold in the livers of the mice that received 1.5 ⫻
105 and 1.5 ⫻ 104 OT-II cells in comparison with those that received 1.5 ⫻ 106 OT-II cells (Fig. 4). This is consistent with the
finding that CD8 T cells are more important than CD4 T cells for
clearance of LM in the spleen, but that CD4 T cells are as protective as CD8 T cells in the liver (42). These results also indicate that
the transfer of fewer numbers of TCR transgenic CD4 T cells and
the corresponding greater proliferation of these cells lead to enhanced protection in the liver. More importantly, mice that received the higher number (1.5 ⫻ 106) of OT-II cells had the same
number of bacteria in the spleen and more bacteria in the liver than
mice that received fewer (1.5 ⫻ 105 OT-II cells). These results
indicate that the decreased proliferation of TCR transgenic CD4 T
cells with increasing numbers of cells transferred cannot be due to
less antigenic stimulation.
High precursor frequencies result in less differentiation of TCR
transgenic CD4 T cells
Because the differentiation of naive T cells into cytokine-producing effectors has been linked to the procession through multiple
rounds of division (13–15), and our results have shown that CD4
T cell division can be greatly limited by unusually high precursor
frequencies, we examined whether high precursor frequencies
limit the extent of CD4 T cell differentiation. We transferred 1.5 ⫻
105 and 1.5 ⫻ 106 CFSE-labeled OVA-specific cells from OT-II or
OT-I mice into congenic recipients and infected them with rLMOVA. On day 8 postinfection, we analyzed the proliferation and
IFN-␥ production of the TCR transgenic cells. We have seen previously that no IL-4⫹ or IL-2 single⫹ (IL-2⫹/IFN-␥⫹ double-positive cells can be detected) CD4 T cells exist in the spleen following i.v. infection with LM (our unpublished results). In mice that
received 1.5 ⫻ 105 OT-II cells, 26.8% of the responding cells
(those that have divided at least once) differentiated into IFN-␥producing cells compared with only 10.7% in mice that received
1.5 ⫻ 106 OT-II cells (Fig. 5A). As expected, 98.1 and 97.2% of
responding OT-I cells produced IFN-␥ in mice that received 1.5 ⫻
105 or 1.5 ⫻ 106 OT-I cells, respectively (Fig. 5B). Therefore,
nearly all of the CD8 T cells that were recruited into division
differentiated into cytokine-producing effectors regardless of initial
precursor frequency. The absolute number of Ag-specific donor
CD4 T cells capable of secreting IFN-␥ was ⬃4.1 ⫻ 104 and 2.1 ⫻
104 in mice that received 1.5 ⫻ 105 and 1.5 ⫻ 106 OT-II cells,
respectively. Thus, twice as many differentiated CD4 T cells were
generated when the initial precursor frequency was 10 times lower.
In contrast, the absolute number of Ag-specific donor CD8 T cells
capable of secreting IFN-␥ was 2-fold less (2.2 ⫻ 106 vs 4.4 ⫻
106) when the initial precursor frequency was 10-fold lower. These
data show that the extent of differentiation of CD4 T cells in this
system is inhibited by abnormally high precursor frequencies, and
that the differentiation of CD4 T cells is inversely related to the
initial precursor frequency.
Discussion
The magnitude of the CD4 T cell response is generally believed to
be lower than that of the CD8 T cell response (7, 22, 43, 44). These
conclusions stem from studies that quantified Ag-specific T cells
using techniques such as intracellular staining for IFN-␥, tetramer
staining, or tracking T cell clones (45). However, these techniques
may underestimate the CD4 T cell response because the percentage of responding CD4 T cells that differentiate into cytokineproducing effectors may be lower than that of responding CD8 T
cells (13, 16), the availability of MHC class II tetramers is limited,
and the response to a few isolated epitopes may not reflect the CD4
T cell response as a whole. For example, for many years, the Agspecific CD4 T cell response to LM infection was considered to be
very weak because the frequency of CD4 T cells responding to all
known LM epitopes was almost too low to study. However, a
recently identified CD4 T cell epitope, LLO190 –201, reacts with
more T cells than any LM-specific CD8 T cell epitope identified to
date (41). In this study, we adoptively transferred naive polyclonal
T cells and compared the CD4 and CD8 T cell responses during a
primary LM infection. We found that both CD4 and CD8 T cells
divide extensively following infection, with nearly all responding
cells in both subsets becoming CFSE negative (dividing seven or
more times). In addition, the absolute number of responding CD4 and
CD8 T cells was similar at the peak of response on day 8 after infection. Combined, these data indicate that the CD4 T cell response to
primary LM infection is as robust as the CD8 T cell response in terms
of the total number of responding cells generated.
The extent of polyclonal CD4 T cell division reported in this
study differed from previous findings that adoptively transferred
TCR transgenic CD4 T cells undergo significantly limited division
in the spleen (11, 12, 46) in comparison with adoptively transferred TCR transgenic CD8 T cells in response to infection (1–3,
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 5. Clonal competition limits the differentiation of CD4, but not
CD8, T cells. The indicated number of OT-II (A) and OT-I (B) cells was
transferred into congenic Thy-1.2 mice. The following day, the recipients
were infected with rLM-OVA. On day 8 postinfection, proliferation of
OT-II (Thy-1.1⫹V␣2⫹CD4⫹) and OT-I (Thy-1.1⫹V␣2⫹CD8⫹) cells was
visualized by loss of CFSE, and differentiation of these cells was analyzed
by intracellular IFN-␥ staining. Numbers indicate the percentage of responding OT-II or OT-I cells that secrete IFN-␥.
3041
3042
INHIBITION OF CD4 T CELL RESPONSE BY CLONAL COMPETITION
of TCR transgenic CD4 T cells to study the response of CD4 T
cells may significantly underestimate the extent of division and
differentiation of the natural response to the same Ag. The effects
of clonal competition on CD4 T cell proliferation and differentiation may need to be considered in designing rational vaccine
strategies, particularly ones that incorporate multiple boosts in
which pools of memory cells may compete with each other.
Acknowledgments
We thank members of the Shen laboratory for discussion and critical reading of the manuscript.
Disclosures
The authors have no financial conflict of interest.
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CD4 T cells. When the precursor frequency of Ag-specific CD4 T
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