Although primary CD8 responses to acute infections are independent
of CD4 help, it is unknown whether a similar situation applies to
secondary responses. We show that depletion of CD4 cells during the
recall response has minimal effect, whereas depletion during the
priming phase leads to reduced responses by memory CD8 cells to
reinfection. Memory CD8 cells generated in CD4+/+ mice
responded normally when transferred into CD4
/ hosts,
whereas memory CD8 cells generated in CD4/ mice mounted
defective recall responses in CD4+/+ adoptive hosts. These
results demonstrate a previously undescribed role for CD4 help in the
development of functional CD8 memory.
Department of Microbiology, University of Pennsylvania School of
Medicine, Philadelphia, PA 19104, USA.
*
To whom correspondence should be addressed at the Department of
Microbiology, University of Pennsylvania School of Medicine, 3610 Hamilton Walk, Philadelphia, PA 19104-6076, USA. E-mail: hshen@mail.med.upenn.edu
The ability to mount
an accelerated response to reinfection is the hallmark of immunological
memory and the basis of vaccination (1). This ability is
mediated by the presence of antigen-specific memory T cells that are
capable of responding more rapidly and in a greater magnitude than
naïve T lymphocytes (1). Primary responses by
naïve CD8 cells to noninflammatory immunogens are known to
require CD4 T cell help (TH), and this help is
thought to occur through activation of antigen-presenting cells (APCs) (2-5) or to involve direct CD40-CD40L interaction between CD4 and CD8 cells (6). However,
the primary CD8 response to infectious agents is often independent of
TH (7-10), and it is hypothesized that recognition of microbial products by Toll-like receptors directly
activates APCs and thus bypasses the need for CD4 help (11).
Relative to the requirements for priming of naïve CD8
responses, little is known about the contribution of TH to the recall response mediated by memory CD8 cells.
To investigate the role of TH in the generation of memory
CD8 cells, we used a heterologous prime/boost regimen that is
particularly effective in boosting antigen-specific memory responses,
thus allowing us to quantitatively measure secondary expansion by
memory CD8 cells (12). CD4+/+ and
CD4/ mice were immunized with a recombinant vaccinia
virus (rVV33) expressing the GP33-41 epitope from lymphocytic
choriomeningitis virus (LCMV) (13-15). At least
30 days later, immunized mice were boosted with a recombinant
Listeria monocytogenes (rLm33) expressing the same epitope
(16). In accordance with previous reports
(9, 10), rVV33 immunization induced comparable
primary CD8 responses and a similar number of GP33-specific CD8 memory
cells in CD4+/+ and CD4/ mice (6.7 × 103 and 5.8 × 103 per spleen,
respectively) (Fig. 1, A and B). Upon
rLm33 boost, GP33-specific memory CD8 cells in CD4 +/+ mice
mounted a strong recall response, expanding to 7.3 × 106/spleen by day 7 after rLm33 infection.
CD4/ mice also mounted a recall response, but the
population of GP33-specific CD8 cells expanded to only 1.1 × 106/spleen, significantly less than in
CD4+/+ mice. This difference was not due to a lower initial
level of memory in CD4/ mice after rVV33-prime
but rather resulted from a greatly reduced expansion of GP33-specific
memory CD8 cells during the recall response in CD4/
mice (184-fold compared to 1088-fold in CD4+/+ mice) (Fig.
1C). These results indicate that CD4 cells play an important role in
supporting a robust recall response by memory CD8 cells upon antigenic
restimulation.
Fig. 1.
Diminished recall response by memory CD8 cells in
CD4/ mice. CD4+/+ (+/+) and
CD4/ (
/) mice were primed with rVV33 alone (rVV33)
or 30 days later were boosted with rLm33 (rVV33 
rLm33).
(A) Detection of GP33-specific cells by intracellular
IFN-
(IFN+/GP33) and MHC/peptide tetramer
(Kb/GP34 and Db/GP33) staining on day 7 after
rLm33-boost (15). Numbers indicate the frequencies as
percentages of splenic CD8 cells. (B) Total numbers of
GP33-specific CD8 cells per spleen. (C) Expansion of
GP33-specific CD8 cells during the recall response, calculated as fold
increase in rVV33 rLm33 mice over rVV33 mice.
[View Larger Version of this Image (63K GIF file)]
To investigate the role of TH during the recall
response, we depleted CD4 cells in vivo either during rLm33-boost or
during rVV33-prime (14). Unexpectedly, depletion of
CD4 cells during rLm33-boost had minimal impact on the recall response
of GP33-specific memory cells (Fig. 2). Instead, depletion of
CD4 cells during the rVV33-prime adversely affected the proliferative potential of memory CD8 cells during subsequent boost with rLm33. GP33-specific cells in these mice expanded only ~200-fold by day 7 after rLm33-boost, compared to >1000-fold expansion in control mice
and mice depleted of CD4+ cells during rLm33-boost. These
results show that CD4 cells play an essential role during the priming
phase for the generation of memory CD8 cells capable of efficient
recall response, whereas TH is not required for
secondary expansion in the boosting phase.
Fig. 2.
Depletion of CD4 cells during priming results
in a defective recall response by memory CD8 cells. CD4+/+
mice were immunized with rVV33 alone (I and II) or immunized with rVV33
and 33 days later boosted with rLm33 (III to V). CD4 cells were
depleted with monoclonal antibodies to CD4 either during rVV33 priming
(II and IV) or during the rLm33 boost (V).
(A) Detection of GP33-specific cells by intracellular
IFN- (IFN/GP33), and MHC/peptide tetramer (Kb/GP34
and Db/GP33) staining on day 7 after rLm33-boost. Numbers
indicate the frequencies as percentages of splenic CD8 cells.
(B) Total numbers of GP33-specific CD8 cells per
spleen.
[View Larger Version of this Image (40K GIF file)]
To more rigorously test the above conclusions, we examined whether
memory CD8 cells primed in CD4+/+ mice could mount a normal
recall response in adoptive CD4/ hosts. LCMV
immunization was used to generate a large population of GP33-specific
memory CD8 cells (17). Purified CD8 cells from
LCMV-immune CD4+/+ mice were transferred into
CD4+/+ and CD4/ mice, which were then
infected with rLm33 (14). Donor CD8 cells mounted a
vigorous GP33-specific recall response that was similar in magnitude in
both CD4+/+ and CD4/ recipients (Fig.
3, A and B). These results further support the conclusion that TH is not required during the
recall response for the rapid expansion of memory CD8 cells.
Fig. 3.
Memory CD8 cells primed in
CD4+/+ mice mount a normal recall response in
CD4/ adoptive hosts, whereas memory CD8 cells generated
in CD4/ mice mount a defective recall response in
CD4+/+ adoptive hosts. (A and B)
Purified CD8+ cells from LCMV-immune (>60 days) Thy1.1
CD4+/+ (+/+) mice were transferred into congenic Thy1.2
CD4+/+ (+/+ +/+) or CD4/ (+/+ /) mice, which were then infected with rLm33. (C to
F) CD8+ cells were purified from Ly5.2
CD4+/+ (+/+) or CD4/ (/) mice that were
previously immunized with LCMV (> 60 days). Purified CD8+
cells, normalized to contain an equal number of GP33-specific cells,
were transferred into congenic Ly5.1 CD4+/+ (+/+) mice,
which were then infected with rLm33. (A and C) Intracellular
IFN- staining of GP33-specific cells on day 0 and 7 after rLM33
infection. Numbers indicate the frequencies as percentages of total
splenic CD8 cells (gated on CD8+ cells). (B and D) Total
numbers of donor GP33-specific cells per spleen at day 0, 4, and 7 after infection (D.P.I.), based on intracellular IFN-
(IFN+/GP33) or MHC/peptide tetramer
(Kb/GP34+ or Db/GP33+)
staining. Triangles: (+/+ +/+); squares: (+/+ /); and
circles: (/ +/+). (E) Total numbers of host GP33-specific CD8
cells per spleen at 0, 4, and 7 D.P.I., as determined by intracellular
IFN- staining. (F) Numbers of rLm33 on day 4 in spleen of mice that
received memory CD8 cells from CD4+/+ (+/+) or
CD4/ (/) mice.
[View Larger Version of this Image (55K GIF file)]
We next examined the ability of memory CD8 cells from
CD4/ mice to mount a recall response in adoptive
CD4+/+ hosts. Purified CD8 cells from LCMV-immune
CD4+/+ and CD4/ mice were transferred into
CD4+/+ recipients. An equal number of GP33-specific CD8
cells were transferred, and these cells engrafted similarly, as was
evident from comparable levels of GP33-specific CD8 cells detected in
recipient mice before rLm33 infection (Fig. 3C). By day 7 after rLm33
infection, GP33-specific memory cells from CD4+/+ mice had
mounted a strong recall response, expanding to a frequency of ~36.6%
of splenic CD8 cells and a total number of 8.8 × 106/spleen (Fig. 3, C and D). In contrast, GP33-specific memory CD8 cells from CD4/ mice mounted a much weaker
recall response, reaching only ~1.0% of splenic CD8 cells and a
total number of 1.3 × 105/spleen. These mice
displayed a higher primary GP33-specific response by the host CD8
lymphocytes (Fig. 3, C and E), presumably compensating for the reduced
expansion by donor memory cells. Furthermore, as a consequence of the
reduced recall response, a lower level of protection against rLm33
infection was observed in these mice, which had 10-fold higher numbers
of rLm33 bacteria on day 4 after infection than mice that received
donor cells from CD4+/+ mice (Fig. 3F). These results
demonstrate that memory CD8 cells generated in the absence of
TH are qualitatively different and are less fit to mount a
vigorous recall response against reinfection, even in the presence of
CD4 cells.
In support of our in vivo results, memory CD8 cells from
CD4+/+ mice proliferated more extensively and produced a
higher level of cytokines than those from CD4/ mice
upon ex vivo antigenic stimulation (fig. S1). Functional impairment of
memory CD8 cells in CD4/ mice was not due to the
inability of these mice to clear infection by rLm33, rVV33, or LCMV
(7, 9, 14). Furthermore,
memory T cells in these mice showed no evidence of recent T cell
receptor activation (fig. S2), indicating that they were not stimulated
by persisting antigens and thus were bona fide resting memory T cells.
Absence of CD4 help has been shown to lead to defective CD8 responses
during chronic viral infection (8,
18-20). Previous studies have also reported reduced
secondary expansion by memory CD8 cells in CD4-deficient mice following
acute viral infections (9, 21), although the
precise reason for this defect has not been examined. Recent studies
have shown that the generation of efficient CD8 memory requires the
presence of CD4 cells during immunization with classically
TH-dependent antigens (6, 22). In
addition, it has been shown that memory CD8 cells primed by viral
infection in the absence of CD4 help mount a defective secondary
response in vitro upon restimulation (22).
Our in vivo data from acute viral and bacterial infections show that
the presence of CD4 cells during the priming phase is critical for
generating functional CD8 memory, whereas TH is not required during the recall response for secondary expansion. These results demonstrate a previously undescribed role for CD4 cells in the
CD8 response to infection that had been thought to be TH independent. It remains to be determined if help in this case involves
direct CD40-CD40L interaction between CD4 and CD8 cells, as suggested
for TH-dependent antigens (6).
Elucidation of the mechanism will help us better understand the
development of CD8 memory and optimize vaccine strategies for inducing
protective immunity.
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Supporting Online Material
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Materials and Methods
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Figs. S1 and S2
13 January 2003; accepted 5 March
2003
10.1126/science.1082305
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