The parameters controlling the generation of robust CD4+ T cell recall responses remain poorly defined. In this study, we compare recall responses by CD4+ and CD8+ memory T cells following rechallenge. Homologous rechallenge of mice immune to either lymphocytic choriomeningitis virus or Listeria monocytogenes results in robust CD8+ T cell recall responses but poor boosting of CD4+ T cell recall responses in the same host. In contrast, heterologous rechallenge with a pathogen sharing only a CD4+ T cell epitope results in robust boosting of CD4+ T cell recall responses. The disparity in CD4+ and CD8+ T cell recall responses cannot be attributed to competition for growth factors or APCs, as robust CD4+ and CD8+ T cell recall responses can be simultaneously induced following rechallenge with peptide-pulsed dendritic cells. Instead, CD4+ T cell recall responses are dependent on the duration of the secondary challenge. Increasing the rechallenge dose results in more potent boosting of CD4+ T cell recall responses and artificially limiting the duration of secondary infection following heterologous rechallenge adversely impacts the magnitude of CD4+ T cell, but not CD8+ T cell, recall responses. These findings suggest that rapid pathogen clearance by secondary CTL following homologous rechallenge prevents optimal boosting of CD4+ T cell responses and therefore have important practical implications in the design of vaccination and boosting strategies aimed at promoting CD4+ T cell-mediated protection.
Following acute infection, Ag-specific CD8+ T cells undergo rapid expansion, dividing 15–20 times and developing CTL function. After clearance of the pathogen, the vast majority (90–95%) of Ag-specific T cells die, leaving behind a population of long-lived memory cells that provide protection from secondary challenges with the same or a related pathogen (1). A major research focus in recent years has been to delineate the nature and timing of the signals that promote the differentiation, function, and longevity of memory T cell populations, as well as their ability to respond to secondary challenges.
Although the activation and differentiation of CD4+ and CD8+ T cells is in many ways similar, important distinctions warrant further study, as they will impact vaccine strategies aimed at inducing and boosting either CD8+ T cell or CD4+ T cell responses. For example, although CD8+ T cells require only a short period (6–24 h) of interaction with Ag to undergo an Ag-independent period of programmed expansion and differentiation (2, 3, 4, 5), CD4+ T cells may require several days of interaction with Ag in vivo for optimal activation and expansion (6, 7). Furthermore, we have recently shown that low-avidity CD4+ T cell responders demonstrate reduced function and survival (8). These results suggest that although CD8+ T cells may rely on Ag for recruitment into the response, once these cells are programmed, differentiation and expansion can be Ag independent (9). CD4+ T cells, on the other hand, conform more to a model in which increasing stimulatory signals progressively drive proliferation, differentiation, and survival (10, 11).
A further distinction between CD8+ and CD4+ T cell differentiation is the stability of their ensuing memory populations. Although CD8+ memory T cells are maintained at stable levels throughout the life of the mouse, in some circumstances CD4+ memory T cells have been noted to decline (12, 13, 14). This decline has led some to question whether these cells can truly be called memory cells, since the population appears to lack the capacity for self-renewal (15). In contrast, CD4+ memory T cells in humans are detectable for up to 75 years after immunization with a half-life similar to CD8+ memory T cells (16). CD8+ and CD4+ memory T cells both depend on the cytokines IL-7 and IL-15 for survival (17, 18), but the other factors that promote long-term CD4+ memory T cell maintenance need to be resolved.
It has also been suggested that CD4+ T cells, whether intrinsically (19) or due to clonal competition (20, 21), are less capable of robust expansion than CD8+ T cells. Competition among CD4+ T cell responders for access to APCs or other resources has been shown to play an important role in regulating cell division. One recent study also found that memory CD4+ T cells displayed limited expansion following secondary challenge due to their production of growth-limiting cytokines even in environments in which Ag was plentiful and the frequency of responders was low (22). These findings suggest that memory CD4+ T cells may be intrinsically limited in the ability to expand upon secondary challenge.
We have repeatedly observed in settings of homologous rechallenge that stimuli sufficient to promote robust secondary expansion of CD8+ memory T cells only weakly promote secondary expansion of CD4+ memory T cells. Because the parameters controlling the effectiveness of CD4+ T cell recall responses have not been well characterized, it is unclear whether these differences are intrinsic, related to the impact of interior intraclonal competition, or due to insufficient access to secondary stimuli. In this study, we found that following heterologous rechallenge with a pathogen sharing only a CD4+ T cell epitope with the primary pathogen, CD4+ memory T cells underwent robust secondary responses comparable to those seen for CD8+ T cells. CD4+ T cell recall responses did not compete with CD8+ T cells for space or resources but rather required a longer stimulatory period following rechallenge. Our findings suggest that homologous rechallenge targeting both CD8+ and CD4+ T cell recall responses results in a shortened infectious time course that, because the pathogen is rapidly cleared by secondary CTL, is sufficient to promote robust secondary CD8+ T cell responses but insufficient to induce optimal CD4+ T cell secondary responses. Although our results also suggest that CD4+ memory T cells may have an intrinsically limited capacity for secondary expansion compared with primary responders, in the context of a prime/boost strategy robust boosting of CD4+ T cell responses to levels comparable to those typically seen for CD8+ T cell responses is possible under appropriate stimulatory conditions. Therefore, these findings have important practical implications for the design of prime/boost vaccination strategies targeting CD4+ T cells.
Materials and Methods
Mice and infections
Six- to 8-wk-old C57BL/6 (B6) and B6.SJL-PtprcaPepcb/BoyJ (B6.SJL) mice were purchased from The Jackson Laboratory. All animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee at the University of Utah. lymphocytic choriomeningitis virus (LCMV)3 Armstrong 53b was grown in BHK cells and titered in Vero cells (23). For primary challenges and heterologous rechallenges, mice were infected i.p. with 2 × 105 PFU. For homologous rechallenges, mice were infected with 2 × 106 PFU i.v. Listeria monocytogenes (Lm)-gp61 (a gift from M. Kaja-Krishna, University of Washington, Seattle, WA, generated using described methods (8, 24, 25)), LM-expressing secreted OVA (Lm-OVA) (26), and wild-type Lm were propagated in brain-heart infusion broth and agar plates. Before infection, the bacteria were grown to log phase and concentration was determined by measuring the OD at 600 nm (OD of 1 = 1 × 109 CFU/ml). For Lm-gp61, mice were injected i.v. with 2 × 105 CFU. For Lm-OVA, mice were given a primary challenge of 2 × 103 CFU and secondary challenges as indicated in the text.
Dendritic cell (DC) immunizations
DCs were expanded in B6 mice with a Flt-3L-secreting B16 mouse melanoma cell line as previously described (27). DCs were enriched to 70–80% purity from spleens and lymph nodes by transient adherence overnight. They were then pulsed with either 0.1 μM LCMV gp33–41 class I-restricted peptide (KAVYNFATC) or 1 μM LCMV gp61–80 class II-restricted peptide (GLKGPDIYKGVYQFKSVEFD) for 2 h in the presence of 1 μg/ml LPS. Mice were injected with 1 × 106 DCs i.v. on days 0, 2, and 4 along with a bystander Lm infection (2 × 103 CFU i.v.) on day 0.
Untouched CD4+ T cells were isolated from the spleens of Lm-gp61 B6 mice (CD45.2+) by incubation with a biotinylated Ab mixture followed by anti-biotin magnetic beads and depletion on a magnetic column per the manufacturer’s recommendations (Miltenyi Biotec). One × 106 cells were injected i.v. into B6.SJL mice (CD45.1+), followed by infection with LCMV.
Peptide restimulation and intracellular cytokine staining
Splenocytes were resuspended in RPMI 1640 containing 10% FBS and supplemented with antibiotics and l-glutamine. Mice were restimulated with 0.1 μM class I-restricted peptide (GP33–41) or 1 μM class II-restricted peptide (GP61–80), as indicated, in the presence of brefeldin A (1 μl/ml GolgiPlug). Cells were stained with cell surface Abs, permeabilized, and stained with cytokine Abs using a kit per the manufacturer’s instructions (BD Biosciences).
Abs and flow cytometry
b. Cell surface Ab staining was done in PBS containing 1% FBS, and intracellular cytokine staining was done as described above. Ab-stained cells were analyzed on a FACSCanto flow cytometer (BD Biosciences) and the results were analyzed using FlowJo software (Tree Star).
Heterologous rechallenge results in potent boosting of CD4+ memory T cells
We directly compared the secondary responses of CD4+ and CD8+ memory T cells following homologous rechallenge. C57BL/6 (B6) mice were infected with LCMV or a recombinant Lm-OVA. At days 8 (peak of the primary response) and 42 (establishment of memory) after infection, immunodominant CD8+ T cell responses (as measured by the GP33–41-specific response for LCMV and the OVA257–264-specific response for Lm-Ova) and CD4+ T cell responses (as measured by the GP61–80-specific response for LCMV and the listeriolysin O (LLO)190–201-specific response for Lm-OVA) in the spleen were measured by ex vivo peptide restimulation followed by intracellular cytokine staining (Fig. 1⇓). Lm-OVA-immune and LCMV-immune mice were then given a high-dose homologous rechallenge, and T cell recall responses were analyzed at the peak of the recall response (day 5 after rechallenge). At rechallenge doses sufficient to potently boost CD8+ T cell responses beyond the initial primary response, secondary CD4+ T cell responses were comparatively poor, with no boosting beyond the levels of the initial primary response (Fig. 1⇓). The poor recall responses observed for CD4+ T cells cannot be simply attributed to delayed secondary responses, as analysis of later times points revealed that day 5 represented the peak of the secondary CD4+ T cell response (data not shown). These findings suggested that in these model systems CD4+ T cells were not boosted at all, as seen in the comparison of peak primary and secondary CD4+ T cell responses (Fig. 1⇓b). We sought to better understand the underlying factors promoting robust CD4+ T cell recall responses, as resolution of these factors could play an important role in the design of vaccination strategies targeting CD4+ T cells.
We formulated several nonexclusive hypotheses to explain these differences. First, memory CD4+ T cell expansion may be intrinsically limited. Second, CD8+ T cells may out-compete CD4+ T cell for resources or access to APCs during secondary expansion. Third, secondary CD4+ T cell expansion may be limited at the population level by intra- or interclonal competition. Lastly, because secondary challenges are rapidly cleared by recall CTL responses, Ag levels or the shortened kinetics of Ag presentation may be insufficient to induce robust and prolonged secondary CD4+ T cell expansion.
To determine whether CD4+ memory T cells were capable of secondary expansion following a more robust secondary challenge that was not rapidly cleared, we adopted a model of heterologous rechallenge. We immunized mice with either LCMV or a recombinant Listeria sharing a single CD4+ T cell epitope with LCMV (Lm-gp61). Following the establishment of memory (>42 days after infection), mice were given a reciprocal rechallenge with either Lm-gp61 or LCMV and their CD4+ T cell recall responses were analyzed at their peak (day 7). Rechallenge of Lm-gp61-immune mice with LCMV resulted in massive expansion of CD4+ memory T cells (Fig. 2⇓, a and b) similar to that seen for CD8+ T cell recall responses following homologous rechallenge. Similarly, rechallenge of LCMV-immune mice with Lm-gp61 resulted in robust, although more modest, expansion of CD4+ memory T cells (Fig. 2⇓, a and b). Importantly, although homologous rechallenge failed to significantly boost CD4+ T cell responses beyond the levels seen after primary challenge, heterologous rechallenge resulted in a significant boosting of the CD4+ T cell response beyond that seen for the primary response. Heterologous rechallenges were characterized by the emergence of IFN-γ-only producing effector cells, whereas the primary response was characterized by the dominance of effector cells capable of producing both IFN-γ and IL-2 upon Ag stimulation (Fig. 2⇓c). These results demonstrate that under the appropriate rechallenge conditions CD4+ memory T cells are capable of equally robust secondary responses as CD8+ memory T cells. At least in the context of rechallenge of an already immune host, CD4+ memory T cells did not display any intrinsic limitation in their ability to divide and accumulate compared with CD8+ memory T cells.
CD4+ and CD8+ T cells do not compete for resources following secondary challenge
We next sought to determine why CD4+ memory T cells failed to respond well following homologous rechallenge but expanded robustly following heterologous rechallenge. Again, we considered several possibilities. Because the heterologous rechallenges specifically and uniquely boosted a CD4+ T cell response without an accompanying CD8+ T cell recall response, we considered the possibility that robust boosting of CD8+ and CD4+ T cells would result in competition for resources such as growth or inflammatory factors such as IFN-γ (28). In this setting, CD4+ T cell recall responses could be generally inhibited and out-competed in the presence of a CD8+ T cell recall response. To address this possibility, we generated a model system of DC immunization in which CD4+ and CD8+ T cells could be rechallenged simultaneously in the presence of a bystander acute infection. We injected mice with B16 cells secreting Flt3L and 2 wk later isolated DCs via transient adherence. DCs were loaded with either the gp33–41 immunodominant LCMV class I-restricted peptide or the gp61–80 immunodominant LCMV class II-restricted peptide and injected into LCMV-immune mice along with a wild-type Lm infection. Injection of either DC subset selectively and potently induced CD8+ or CD4+ T cell recall responses, respectively, while injection of both DC subsets simultaneously induced both CD8+ and CD4+ T cell recall responses in the same host (Fig. 3⇓). These findings confirm that even robust CD8+ T cell recall responses do not out-compete CD4+ T cell recall responses for resources and growth factors. However, they leave open the possibility that large numbers of responding CD8+ T cells could prevent sufficient access to APCs by CD4+ T cells.
Increasing the rechallenge stimulus results in enhanced CD4+ T cell recall responses
We next hypothesized that the discrepancies between CD4+ and CD8+ T cell recall responses following recall responses could be due to differential stimulatory requirements. Because CD8+ T cell recall responses rapidly cleared secondary challenges in these settings, one possible consequence was an insufficient duration or potency of stimulus to induce robust CD4+ T cell recall responses. This might involve an increased reliance on Ag, as has been demonstrated for primary CD4+ T cell responses, or an increased need for inflammatory stimuli that are limited by rapid clearance mediated by CD8+ T cell recall responses. As an initial approach to address this possibility, we gave Lm-OVA-immune mice a homologous rechallenge with increasing doses of Lm-OVA. CD8+ T cell recall responses directed toward OVA257–264 were potently induced following rechallenge with 1 × 105 CFU of Lm-OVA, and increasing the rechallenge dose beyond these levels did not significantly enhance the magnitude of this response (Fig. 4⇓). In contrast, increasing the rechallenge dose progressively and significantly boosted CD4+ T cell recall responses directed toward LLO190–201 (Fig. 4⇓). These results demonstrate that rechallenge doses sufficient to recruit a maximally potent CD8+ T cell recall response only weakly recruit CD4+ T cell recall responses and suggest that CD4+ memory T cells are tightly regulated by exposure to the secondary stimulus. CD8+ T cells are also likely to be tightly regulated by access to Ag, and these findings do not rule out the possibility that MHC class I-restricted responses are more readily induced at lower infectious doses due to wider distribution of MHC class I expression and Ag presentation. Conversely, DCs are required for initiating robust recall responses (29), suggesting that at least during the initiation of secondary responses, Ag presentation to CD8+ and CD4+ memory T cells is similar. In either case, our findings suggest that in the context of an in vivo secondary challenge CD4+ memory T cell recall responses display a higher activation threshold as it relates to infectious dose than do CD8+ T cells.
CD4 recall responses require a longer stimulatory period than CD8 recall responses
As a second approach to assessing the impact of the strength or duration of the secondary stimulus in controlling CD4+ T cell recall responses, we artificially limited the duration of the secondary infection following heterologous rechallenge of either CD4+ or CD8+ T cell recall responses. We rechallenged LCMV-immune mice with either Lm-gp61 to boost CD4+ T cell recall responses or a recombinant Listeria secreting the LCMV gp33–41 peptide (Lm-gp33) to boost CD8+ T cell recall responses. Mice were treated with ampicillin to limit the course of infection at 12, 24, or 48 h after infection. Ampicillin treatment in our hands resulted in bacterial clearance within 24 h (data not shown). Furthermore, using previously described methods (7), we assessed Ag presentation by transferring LCMV glycoprotein-specific CFSE-labeled TCR-transgenic T cells (P14 T cells following Lm-gp33 infection or SMARTA T cells following Lm-gp61 infection) into ampicillin-treated mice at various time points after treatment. TCR-transgenic T cells failed to dilute CFSE when transferred 48 h after ampicillin treatment, indicating minimal Ag presentation (data not shown).
Although CD8+ T cell recall responses neared the maximal response with ampicillin treatment as early as 24 h after infection, CD4+ T cell responses were significantly lower even when ampicillin treatment began as late as 48 h after infection (Fig. 5⇓). In comparison, primary responses analyzed at the same time demonstrated a highly similar pattern for both CD8+ (gp33–41-specific) and CD4+ (gp61–80-specific) T cells (data not shown), agreeing with a previously published report (7). These results are similar to previous studies of primary CD4+ and CD8+ T cell responses and again suggest that CD4+ T cell recall responses require an extended stimulatory period as compared with CD8+ T cell recall responses.
Secondary proliferation of CD4+ memory T cells is intrinsically limited
Lastly, we sought to determine the extent to which clonal competition could inhibit the generation of robust CD4+ T cell recall responses. Competition among activated monoclonal CD4+ T cells has been shown to prevent adequate access to Ag, resulting in blunted CD4+ T cell responses (20). Although the CD4+ T cell recall responses analyzed in this study are polyclonal, we hypothesized that competition for access to the same Ag when dose is limiting (as during homologous rechallenge) could prevent optimal CD4+ T cell secondary expansion. Conversely, when dose is not limiting (as during heterologous rechallenge), secondary expansion could proceed unimpeded. However, even under the best stimulatory conditions Ag-specific recall responses represent only a 50- to 100-fold expansion over memory levels. In contrast, primary expansion of CD4+ T cells to LCMV infection has been estimated at >10,000-fold, as based on enumeration of naive precursors (30, 31). The factors that limit secondary expansion are unclear, particularly given that the kinetics of infection following heterologous boosting of CD4+ T cell recall responses are similar to that of the primary infection.
We hypothesized that secondary expansion of CD4+ T cells was limited by competition for Ag or resources. To address this issue, we transferred CD4+ T cells from Lm-gp61-immune B6 (CD45.2+) mice into naive B6.SJL (CD45.1+) congenic hosts, followed by challenge with LCMV. The size of the transfer was designed to generate precursor frequencies of gp61–80-specific CD4+ memory cells approaching endogenous levels of naive precursor cells. Based on our analysis of the frequency of Ag-specific IFN-γ-producing memory cells in the initial transfer, as well as the measurement of the “take” of the initial transfer 1 day later, we estimated a total of ∼600–800 gp61–80-specific memory cells in the spleens of secondary hosts before challenge. The frequency of naive endogenous responders specific for this epitope has been estimated at 100–200 (31). This system therefore allowed us to analyze primary and secondary responses in the same host following LCMV infection while mitigating the effects of competition for Ag among the two populations. We challenged mice with LCMV and analyzed secondary responses in the spleen 8 days later. Following LCMV heterologous rechallenge of Lm-gp61-immune hosts, CD4+ T cell recall responses expanded robustly (60–70-fold; Fig. 6⇓, a and b). Although LCMV rechallenge of B6.SJL hosts that had received CD4+ memory T cells also resulted in secondary expansion (80–90-fold), it only marginally exceeded the expansion seen following rechallenge of Lm-gp61-immune hosts (Fig. 6⇓, a and b). These results suggested an intrinsic limit to the recall potential of CD4+ memory T cells even in the presence of an abundant secondary stimulus. To rule out the possibility that secondary responses were peaking earlier, we repeated the experiments and analyzed recall responses at day 5 and day 8 after challenge. CD4+ memory T cells did not display any significant differences in their recall responses at day 5 or day 8, expanding to a maximum of ∼60-fold. In contrast, primary responders, based on an estimated precursor frequency of 200, expanded almost 1,000-fold by day 5 and >10,000-fold by day 8 (Fig. 6⇓c). These findings suggest that secondary responses by CD4+ memory T cells are intrinsically curtailed and parallel another recent study suggesting an intrinsic limit in secondary CD4+ T cell expansion (22). Although our previous results indicate that enhancing the secondary stimulus can propel a CD4 recall response toward its upper limits, these findings clearly indicate that an upper limit does indeed exist. It will be critical in future studies to define the parameters that both promote and regulate secondary expansion of CD4+ memory T cells.
Our findings highlight key differences between the stimulatory requirements for inducing secondary expansion of CD4+ and CD8+ T cells and parallel differences seen in the generation of primary responses. In those earlier studies, an extended infectious period (as compared with that needed for CD8+ T cells) was required for the full expansion of Ag-specific CD4+ T cells (7). Furthermore, expansion in the setting of primary stimulation was found to be dependent on the continued presence of Ag (6). Importantly, whereas the size of the response was altered, the differentiation of effector function appeared to be independent of the duration of infection (7). In the present study, we found that secondary CD4+ T cell responses are similarly dependent on the duration of infection for their secondary expansion. Therefore, in settings where Ag is cleared quickly, such as following a homologous rechallenge, CD8+ T cell recall responses are efficiently generated, whereas CD4+ T cell recall responses are poor. In contrast, heterologous rechallenge with a pathogen sharing only a CD4+ T cell epitope results in an infectious time course similar to a primary challenge. We conclude, therefore, that the differing magnitude of the CD4+ T cell recall response induced following heterologous vs homologous rechallenge reflects the pace of CD8+ T cell-mediated pathogen clearance.
One important unknown arising from these studies is the mechanism behind the differential requirements for robust secondary expansion. CD4+ memory T cells have a demonstrably lower activation threshold at the level of TCR signaling than their naive counterparts (32). However, CD4+ T cells may have an intrinsically lower proliferation rate than CD8+ T cells (19), and their clonal expansion, at least during the primary response, is mildly delayed compared with CD8+ T cells (31, 33). Although it is difficult to directly compare Ag sensitivity of CD4+ and CD8+ T cells, one possibility is that CD4+ T cells in general have a higher activation threshold for activation and/or a continued dependence on Ag for clonal expansion. From the standpoint of preventing autoreactive or immunopathologic responses, such tight regulation of CD4+ T cells plays an important immunoregulatory function. On the other hand, the dependence by CD4+ memory T cells on an extended infectious period to mount robust recall responses may reflect a role for inflammatory or costimulatory factors in driving their continued expansion independent of the continued presence of Ag. For example, type I IFNs drive clonal expansion of Th1 cells in some settings (34), while OX-40 costimulation plays a role in promoting CD4+ T cell recall responses (35). Whether CD4 expansion is driven by Ag availability or nonspecific access to inflammatory, growth, or costimulatory factors, intra- and/or interclonal competition likely plays an important role in regulating the magnitude of the CD4+ T cell recall response. High frequencies of clonal populations of T cells can also out-compete CD8+ T cell responses to the same epitope. To a much lesser extent, large frequencies CD8+ T cell responders may inhibit the response to other class I-restricted epitopes (36, 37, 38, 39). Although there is no current evidence that large numbers of CD8+ T cells can efficiently out-compete class II-restricted T cell responses, our findings do not rule out the possibility that in the presence of robust CD8+ recall responses, CD4+ memory T cells do not have sufficient access to APCs to induce optimal recall responses.
Although our findings suggest an important role for the duration of secondary infection in promoting secondary CD4 responses, they do not rule out a role for the strength of the initial antigenic stimulus in driving secondary responses. Ag dose is extremely important in the differentiation of CD4+ T cells, and we have recently found evidence for the role of Ag dose in CD4+ effector and memory T cell differentiation (8). We hypothesize that both the Ag dose and the duration of infection play important roles in primary and secondary CD4 differentiation. Because disrupting the duration of Ag presentation during secondary responses impairs the secondary CD4+ T cell response, we have concluded that the duration of infection is one factor in the induction of robust CD4 recall responses. These findings correspond to the original homologous rechallenges in which the secondary infection is shortened due to rapid CTL-mediated clearance. But it is possible, even likely, that increasing the Ag dose during the early stages of infection could compensate for the shortened infectious period. Also, these results do not distinguish the relative roles of continued Ag presentation and the inflammatory environment induced by the infection.
A striking difference between the poor CD4+ T cell recall responses induced by homologous rechallenge and the robust recall responses induced by heterologous rechallenge is the cytokine-producing profile. In particular, heterologous rechallenge promotes the accumulation of CD4+ effector T cells capable of producing IFN-γ but not IL-2. Recent studies have demonstrated that the CD4+ memory T cells capable of the most robust secondary protection maintain the ability to produce IFN-γ, TNF-α, and IL-2, whereas the absence of IL-2- or TNF-α-producing capability signals a reduced recall capacity (40, 41). An important question will be the extent to which the observed skewing toward IFN-γ-only producers following heterologous challenge impacts the makeup and functionality of ensuing memory populations.
One recent study suggested that CD4+ memory cells are intrinsically less capable of clonal expansion than their naive counterparts (22). We also find that CD4+ memory T cell are limited in their expansion even following heterologous challenge and when present at frequencies approaching naive responders. Although limited exposure to Ag during homologous rechallenge limits that ability of CD4+ T cells to reach the upper limit of their recall potential, we also suggest that there is an upper limit. These findings are not mutually exclusive but represent two aspects that regulate CD4 recall responses. Although our data suggest that the duration of infection may impact the magnitude of secondary expansion, it does not suggest that there is no upper limit to that expansion. Rather, we find that secondary expansion by CD4+ T cells is intrinsically controlled. Naive responders expanded 10,000- to 20,000-fold following infection with LCMV, while memory responders in the same host present at similar frequencies expanded 50- to 100-fold, a similar level of expansion seen following heterologous rechallenge but severely curtailed when compared with the primary response in the same host. These findings lead to important questions regarding the biology of CD4+ T cell recall responses. For instance, is there a combined limit of expansion following primary activation and subsequent boosts, or does the proliferative potential of the ensuing memory population “reset” after each challenge? How do successive boosts impact the protective function of CD4+ memory T cells? And, lastly, in the absence of competition for Ag or resources, what are the mechanisms that control the magnitude of secondary CD4+ T cell responses?
Importantly, because our findings suggest that the optimal conditions for boosting CD4+ or CD8+ T cell responses differ substantially, they have relevance for the design of vaccination strategies aimed at specifically boosting CD4+ T cell responses. Vaccines that simultaneously attempt to boost CD4+ and CD8+ T cell responses may be inefficient in promoting optimal secondary expansion of CD4+ memory T cells due to rapid clearance of Ag by CTL recall responses.
The authors have no financial conflict of interest.
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 the National Institutes of Health (K22AI071112) and the Department of Pathology at the University of Utah.
↵2 Address correspondence and reprint requests to Dr. Matthew A. Williams, Department of Pathology, University of Utah, 15 North Medical Drive East, Salt Lake City, UT 84112. E-mail address:
↵3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; Lm, Listeria monocytogenes; Lm-OVA, Lm-expressing secreted OVA; DC, dendritic cell; LLO, listeriolysin O.
- Received January 30, 2009.
- Accepted June 22, 2009.
- Copyright © 2009 by The American Association of Immunologists, Inc.