Recall Responses by Helpless Memory CD8⁺ T Cells Are Restricted by the Up-Regulation of PD-1

Mice and virus infections

C57BL/6 (B6) and congenic B6-Ly5.2-Cr (which are Ly5.1/CD45.1⁺) mice were purchased from The National Cancer Institute (Bethesda, MD). CD40^−/− and TRAIL^−/− mice were generously provided by Dr. Randolph J. Noelle (Dartmouth Medical School, Lebanon, NH) and Amgen, respectively, and were used with permission. The Animal Care and Use Program of Dartmouth College approved all animal experiments. For infections, mice were infected with 10³ PFU of VV-WR (originally obtained from Dr. William R. Green, Dartmouth Medical School, Lebanon, NH) through the i.n. route, and loss of body weight was measured as previously described (32). Murine gammaherpesvirus-68 (MHV-68) virus (clone G2.4) was originally obtained from Dr. A. A. Nash (University of Edinburgh, Edinburgh, U.K.) and was propagated and titered as previously described (32). A recombinant vaccinia virus expressing the open reading frame (ORF)6_487–495/D^b epitope of MHV-68 (rVV-ORF6) was obtained from Dr. Peter Doherty (St. Jude Children’s Research Hospital, Memphis, TN). For MHV-68 infection, 400 PFU was given i.n.

Ab treatments

For depletion of CD4⁺ T cells, mice were treated with 500 μg of anti-CD4 (GK1.5) on days −1 and 0 of infection, followed by 250 μg on day 3 postinfection (p.i.) and twice weekly thereafter, resulting in <0.1% CD4⁺ T cells (data not shown). Control mice were either untreated or given rat IgG (R-IgG; Jackson ImmunoResearch Laboratories). No differences in T cell responses were observed with R-IgG injection (data not shown). The hybridoma producing anti-CD40 mAb (FGK115) was kindly provided by Dr. Randolph J. Noelle. Anti-CD40 mAb (100 μg) was injected on days 1 and 7 p.i., and 500 μg of anti-PD-1 (RMP1–14) was injected on days 0, 2, and 4 during challenge. Injection of IL-2 immune complexes has been described previously (32, 33). Briefly, a mixture of 50 μg of anti-IL-2 mAb (S4B6) and 1.5 μg of murine IL-2 (eBioscience) was injected i.p. daily from day 0 to 4 postchallenge.

MHC/peptide tetramer, Ab, intracellular staining, and flow cytometric analysis

Single-cell suspensions of spleen and lung lymphocytes were prepared as described previously (32). MHC/peptide tetramer for the VV-WR epitope B8R_20–27(TSYKFESV)/K^b or ORF6_487–495(AGPHNDMEI)/D^b conjugated to allophycocyanin was obtained from the National Institutes of Health Tetramer Core Facility (Emory University, Atlanta, GA). Cells were stained for 1 h at room temperature and were further stained with PerCP-conjugated anti-CD8α (53-6.7; BD Pharmingen) along with Abs against surface markers (32). For PD-1 expression, PE-conjugated anti-PD-1 (J43; BD Pharmingen) was used. Staining with PE-conjugated anti-CTLA-4 (UC10-4F10-11; BD Pharmingen) and anti-Bcl-2 (BD Pharmingen) expression was performed at 4°C for 30 min after staining with tetramer, anti-CD8, and FITC-conjugated anti-CD45.2 (clone 104; eBioscience), followed by cell fixation with 1% formaldehyde (Ted Pella) and permeabilization with staining buffer containing 0.5% saponin (Sigma-Aldrich). Annexin V staining was performed after tetramer and CD45.2, CD8 staining according to the manufacturer’s protocol (BD Pharmingen). Intracellular cytokine staining was performed after restimulation of splenocytes with 1 μg/ml peptide, 10 U/ml IL-2, and 5 μg/ml brefeldin A for 5 h at 37°C. IFN-γ production in control wells with no peptide was subtracted as background, as previously described (32).

Adoptive transfer and challenge experiments

Similar to our previous study (32), CD8⁺ T cells were purified (typically >95% purity) from spleens of VV-WR-infected mice (40+ days p.i.), and equal numbers of CD8⁺ T cells (2 to 4 × 10⁶ cells) were injected into congenic B6-CD45.1 (B6-Ly5.2-Cr) mice i.v. Before transfer, cells from each group were stained to determine the percentage of CD8⁺ T cells and tetramer⁺ cells (similar frequencies were observed in all experiments; see Fig. 1⇓C), and the data were used to determine the exact number of virus-specific memory CD8⁺ T cells injected into each individual recipient. One day after transfer, mice were challenged i.p. with 2 × 10⁶ PFU of VV-WR. Spleens were harvested 5 days postchallenge and stained with VV-B8R-tetramer, anti-CD8, and anti-CD45.2, and the total number of donor-derived VV-specific CD8⁺ T cells was calculated. To calculate fold expansion of donor-derived VV-specific CD8⁺ T cells in each individual recipient, the total number of virus-specific memory CD8⁺ T cells 5 days postchallenge was divided by the total number of virus-specific CD8⁺ T cells initially transferred. Four to seven recipients per group were used in each experiment.

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FIGURE 1.

CD4 help does not affect the initial disease and CD8⁺ T cell kinetics during VV infection. A, Weight loss after infection was measured. Numbers indicate surviving mice. B and C, The VV-B8R-specific CD8⁺ T cell response was measured by tetramer staining on day 10 (B) and day 69 (C) p.i. Total numbers of CD8⁺tetramer⁺ cells in each organ are graphed. Open bars indicate undepleted; black bars, CD4-depleted. Representative data from two independent experiments with four mice per group are shown. Error bars indicate SEM.

For experiments involving MHV-68-specific memory CD8⁺ T cells, CD8⁺ T cells were purified from infected mice, and equal numbers of ORF6-specific CD8⁺ T cells (10⁴ cells) as determined by tetramer staining were transferred into naive congenic recipients. We chose to transfer equal number of tetramer⁺ cells because significantly higher frequencies of ORF6-specific memory CD8⁺ T cells were observed in CD4-depleted mice compared with untreated mice (data not shown), as opposed to VV-WR infection, where similar numbers of memory cells are observed (see Fig. 1⇑C). One day after transfer, recipients were challenged with 2 × 10⁶ PFU of rVV-ORF6 i.p., and the expansion of ORF6-specific CD8⁺ T cells was calculated as described above using ORF6 tetramers (32).

Statistical analysis

Values of p were calculated using Student’s t test, and p < 0.05 was considered significant.

CD4 help in early viral disease and the virus-specific CD8⁺ T cell response

Intranasal infection by VV-WR represents a natural route of infection by poxviruses and provides a model for studying CD8⁺ T cell responses to acute cytopathic infection through the mucosal route (32, 34). During the infection a robust CD8⁺ T cell response is generated, allowing us to study the generation of memory CD8⁺ T cells without the use of transgenic T cells, which may skew the results of our studies (35, 36). We depleted CD4⁺ T cells by repeated injection of anti-CD4 Ab (GK1.5) to study the effect of CD4 help on CD8⁺ T cell responses specific to the immunodominant epitope of VV-WR, B8R_20–27 (referred to as VV-specific CD8⁺ T cells). Infection with a sublethal dose (10³ PFU) of VV-WR through the i.n. route results in severe virulence, which can be observed by significant weight loss, followed by recovery after day 8 p.i. (32). We observed no role for CD4⁺ T cells in the initial disease caused by the infection, as CD4 depletion did not have any significant effect on the severity of the disease (Fig. 1⇑A).

In most viral infection models, CD4 help does not affect the primary virus-specific CD8⁺ T cell response, but in some cases help is required for maintaining the memory population. Therefore, we examined the VV-specific CD8⁺ T cell response in the lung and the spleen using MHC/peptide tetramers. CD4⁺ T cells did not affect the primary virus-specific CD8⁺ T cell response in the lung and the spleen (Fig. 1⇑B), which occurs at day 10 in this model (32). Thus, CD4⁺ T cells play no role in the early events that occur during this infection. At 2 mo postinfection, we observed a slight decrease in the frequency of virus-specific memory CD8⁺ T cells in the lung of CD4-depleted mice (data not shown); however, the total numbers of virus-specific memory CD8⁺ T cells in the spleen and lungs were unaffected by the absence of CD4⁺ T cells (Fig. 1⇑C). Overall, CD4 help does not affect the kinetics of the virus-specific CD8⁺ T cell response during VV-WR infection.

Helpless memory CD8⁺ T cells are severely impaired in function

Numerous studies have provided evidence for a crucial role of CD4 help in memory CD8⁺ T cell differentiation (7). Thus, although the kinetics of the virus-specific memory CD8⁺ T cell response was unaffected by the absence of help, we suspected that the helpless memory cells may be functionally impaired. First, we investigated the phenotype of VV-specific memory CD8⁺ T cells in the spleen and the periphery (lungs) in the absence of CD4 help. Helpless memory cells in the spleen expressed lower levels of CD44, CD127 (IL-7Rα), CD122 (IL-2/15Rβ), and CD27 as measured by mean fluorescent intensity (MFI), and there were lower percentages of CD127^high, CD62L^high, and CD27^high cells (Fig. 2⇓A). The helpless memory cells in the lung also expressed lower levels of CD44 and CD122 (Fig. 2⇓B). We observed minimal expression of CD25 and CD69 in the untreated and CD4-depleted groups in both organs, indicating that the cells had a resting phenotype (data not shown). Second, we tested the ability of helpless memory cells to produce cytokines in response to restimulation by intracellular cytokine staining. In the absence of CD4 help, memory cells produced lower levels of IFN-γ, as measured by MFI (Fig. 2⇓C). The frequency of IFN-γ-producing CD8⁺ T cells was slightly reduced, but was not statistically significant (data not shown). Furthermore, lower frequencies of IFN-γ-producing memory cells produced TNF-α and IL-2 (Fig. 2⇓C).

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FIGURE 2.

Helpless VV-specific memory CD8⁺ cells are impaired in function. A, Phenotype of VV-B8R-specific memory CD8⁺ cells in undepleted and CD4-depleted mice were analyzed at day 69 p.i. by tetramer staining. Plots are gated on CD8⁺tetramer⁺ cells. B, Phenotype of lung-resident memory cells. C, Cytokine production by VV-specific memory cells was examined by intracellular cytokine staining. Plots are gated on CD8⁺ cells. Numbers indicate percentage of CD8⁺ T cells producing IFN-γ (left), or percentage of IFN-γ⁺ cells that produce TNF-α (middle) or IL-2 (right). D, The recall response of helped and helpless memory cells were analyzed by an adoptive transfer approach (see Materials and Methods for details). CD8⁺ T cells were purified from undepleted or CD4-depleted VV-infected mice 4 mo p.i. and were transferred into naive congenic (CD45.1⁺) recipients. One day later, recipients were challenged i.p. with 2 × 10⁶ PFU of VV-WR, and 5 days postchallenge expansion of donor-derived VV-specific memory CD8⁺ T cells was analyzed by tetramer staining before and after (lower panel) challenge. E, Results of the adoptive transfer experiment. The total numbers of donor-derived VV-specific CD8⁺ T cells analyzed by tetramer staining before transfer and 5 days postchallenge are graphed. Fold expansion of tetramer⁺ cells was calculated for each recipient, and the number shown indicates the average fold expansion for each group. Each circle (○, undepleted → B6; •, CD4-depleted → B6) represents an individual recipient. Representative plots from two to three independent experiments using three to four mice per group are shown. In D, four donor mice per group and six to seven recipients mice per group were used. ∗, p > 0.05 and ∗∗, p > 0.01.

Finally, we measured the ability of the helpless memory cells to elicit a robust recall response by utilizing an adoptive transfer approach used previously (Fig. 2⇑D) (32). CD4 help is required for the induction of neutralizing Abs; thus, directly rechallenging helped and helpless mice would result in different doses of virus infecting the host. Therefore, we purified CD8⁺ T cells from infected helped or helpless mice and transferred them into naive congenic (CD45.1⁺) hosts, which were challenged 1 day later with 2 × 10⁶ PFU of VV-WR i.p. Before transfer, we calculated the exact number of virus-specific CD8⁺ T cells transferred into each host by tetramer staining (see Materials and Methods). Five days later the recall response was analyzed in the spleen through tetramer staining, and the total number of donor-derived virus-specific CD8⁺ T cells was calculated. This approach allows us to accurately measure the fold expansion of virus-specific CD8⁺ T cells during recall in vivo in each individual animal, through dividing the total number of donor-derived virus-specific CD8⁺ T cells 5 days postchallenge by the total number initially transferred. VV-specific memory CD8⁺ T cells that received CD4 help expanded an average of 39-fold upon challenge, while helpless memory cells expanded on average ∼10-fold (Fig. 2⇑E). Thus, VV-specific memory CD8⁺ T cells that developed in the absence of CD4 help were severely impaired in their ability to mount a recall response upon viral challenge (Fig. 2⇑E). Although the magnitude of reduction varied between experiments, we consistently observed reduction of the recall response by helpless memory cells in all experiments, typically 2- to 4-fold, similar to numbers reported in other models (3, 11, 22, 30, 37). The data clearly demonstrate the requirement of CD4 help for memory CD8⁺ T cell differentiation during acute VV infection.

During lymphocytic choriomeningitis virus infection, lack of CD4 help or CD40L-CD40 interactions leads to viral resurgence at ∼2 mo postinfection, which causes deterioration of memory CD8⁺ T cell function (38). However, we found no evidence of viral persistence in CD4-depleted mice (data not shown), suggesting that the defects observed were a direct effect of CD4 help and not an indirect effect of CD8⁺ T cell exhaustion caused by viral persistence.

Role of CD40 signaling in helping memory CD8⁺ T cell differentiation

The requirement for CD4 help in memory CD8⁺ T cell differentiation has been documented in various infection models, yet the timing of help and the signals that mediate help have been a subject of debate (7, 8). Therefore, we utilized our adoptive transfer system to investigate the required timing and the molecular nature of CD4 help for VV-specific memory CD8⁺ T cells to acquire the ability to mount a robust recall response. First, we tested whether CD4 help is required during priming, by injecting the anti-CD4 mAb only on days −1, 0, and 3. VV-specific memory CD8⁺ T cells from mice that received early CD4 depletion were defective in mounting a recall response (Fig. 3⇓A). The data support the requirement for CD4⁺ T cells during priming, although they do not rule out an additional role of CD4 help during the maintenance phase. Next, due to the importance of CD40L-CD40 interactions in mediating CD4 help (14, 15, 16), we investigated the role of CD40 signaling in memory cell development by transferring VV-specific memory CD8⁺ T cells from infected B6 or CD40^−/− mice. Upon adoptive transfer and challenge with VV-WR, memory CD8⁺ T cells that developed in CD40^−/− mice showed a significantly reduced recall response (Fig. 3⇓B). Therefore, during VV-WR infection CD40 signaling significantly impacts memory CD8⁺ T cell differentiation.

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FIGURE 3.

Functional VV-specific memory cells require CD4 help during priming and CD40 signaling. A, Mice were infected with VV-WR and were untreated or CD4-depleted during priming (days 1, 0, and 3). One hundred days p.i., the recall response of VV-specific memory CD8⁺ T cells was analyzed by adoptive transfer experiments described in Fig. 2 (○, → B6; •, early CD4-depleted → B6). B, B6 or CD40^−/− mice were infected with VV-WR and the recall response of VV-specific memory CD8⁺ T cells was analyzed by adoptive transfer and challenge (○, B6 → B6; •, CD40^−/− → B6). Representative data from two independent experiments are shown using four donors per group and four to eight recipients per group. Each circle represents an individual recipient. Fold expansion of tetramer⁺ cells was calculated for each recipient, and the number shown indicated the average fold expansion for each group. ∗∗, p > 0.01.

The requirement for CD4 help during priming and CD40 signaling prompted us to assess whether providing CD40 stimulation during priming in the absence of CD4⁺ T cells can reverse the defects of helpless memory cells. Agonistic anti-CD40 mAb was injected into CD4-depleted mice at days 1 and 7 p.i., and at 49 day p.i., memory CD8⁺ T cells were analyzed for their phenotype (Fig. 4⇓). The aberrant expression of CD44, CD127, CD27, and CD62L observed in CD4-depleted mice was restored by CD40 signaling during priming. Furthermore, the defects in cytokine production, particularly TNF-α and IL-2, were restored by CD40 ligation back to levels similar to memory cells that received CD4 help (Fig. 5⇓, A and B). IFN-γ production was also slightly increased, although complete restoration of production of this cytokine was never observed. Finally, the defect in the secondary response by the helpless cells was reversed by administering the agonistic anti-CD40 mAb during priming (Fig. 5⇓C). The results show that CD40 stimulation during priming can rescue helpless CD8⁺ T cell memory differentiation, and may indicate a role for CD40 in CD4 help. However, administering CD40 stimulation during priming slightly enhanced recall responses of helped memory CD8⁺ T cells in undepleted mice (Fig. 5⇓D), implying that CD40 signaling may have a nonoverlapping role with CD4⁺ T cell help.

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FIGURE 4.

Providing CD40 stimulation during priming restores phenotypic changes of helpless memory cells. Mice were infected with VV-WR and were undepleted, CD4-depleted/R-IgG treated, or CD4- depleted/anti-CD40 treated (days 1 and 7). A, VV-specific memory CD8⁺ T cells were analyzed for expression of phenotypic markers by tetramer staining on day 49 p.i. Plots are gated on CD8⁺tetramer⁺ cells. B, Graphed presentation of A. Open bars indicate undepleted; gray bars, CD4-depleted/R-IgG treated; black bars, CD4-depleted/anti-CD40 treated. Representative data from two independent experiments with three to four mice per group are shown. Error bars indicate SEM. ∗, p > 0.05 and ∗∗, p > 0.01.

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FIGURE 5.

Providing CD40 stimulation during priming boosts memory cell function in the absence of CD4 help. Mice were infected with VV-WR and were undepleted, CD4-depleted/R-IgG treated, or CD4-depleted/anti-CD40 treated (days 1 and 7). A, Cytokine production by VV-specific memory CD8⁺ T cells was analyzed by intracellular cytokine staining. Plots are gated on CD8⁺ cells. Numbers indicate IFN-γ MFI of CD8⁺ T cells producing IFN-γ (left) or percentage of IFN-γ⁺ cells that produce TNF-α (middle) or IL-2 (right). B, Graphed representation of A. Open bars indicate undepleted; gray bars, CD4-depleted/R-IgG treated; black bars, CD4-depleted/anti-CD40 treated. Representative data from two independent experiments with three to four mice per group are shown. Error bars indicate SEM. ∗, p > 0.05 and ∗∗, p > 0.01. C, The recall response by VV-specific memory CD8⁺ T cells was analyzed by adoptive transfer and challenge. Treatments of donor mice are indicated below. Numbers indicate average fold expansion of VV-specific memory cells for each group. Each circle represents an individual recipient. D, Recall response of helped (undepleted) memory cells that received R-IgG or anti-CD40 during priming is graphed. Fold expansion of transferred (CD45.2⁺) memory cells is shown (error bars indicate SEM). Representative plots are shown from two independent experiments consisting of three to four mice per group. In C and D, four donor mice per group and four to six recipients per group were used.

The helpless defect is independent of TRAIL

Although our data demonstrate that CD4 help is required for VV-specific memory CD8⁺ T cells to acquire the ability to mount a robust recall response, the molecular mechanism of why helpless memory cells elicit defective recall responses remains elusive. Evidence provided by Janssen et al. suggested that the up-regulation of TRAIL by helpless memory cells upon recall leads to increased cell death and an impaired recall response (30). Therefore, we hypothesized that the absence of CD4 help leads to increased cell death of memory CD8⁺ T cells during recall. VV-specific memory CD8⁺ T cells from undepleted, CD4-depleted/R-IgG-treated, or CD4-depleted/anti-CD40-treated mice were challenged with VV-WR upon adoptive transfer into naive recipients (as described in Fig. 5⇑C), and 5 days later, donor tetramer⁺ cells were analyzed for cell death by annexin V staining. No differences in the percentage of virus-specific CD8⁺ T cells undergoing cell death were observed among the three groups (Fig. 6⇓A). Furthermore, we found no difference in the expression of the antiapoptotic molecule Bcl-2 by tetramer+ cells (Fig. 6⇓A). Thus, the data imply that the reason for the impaired recall response by helpless memory cells is not due to increased cell death. We next examined whether TRAIL deficiency could reverse the defective recall response by helpless memory cells, as previously shown (30, 37). As shown in Fig. 6⇓B, VV-specific memory CD8⁺ T cells from CD4-depleted mice were impaired in their recall response, and this defect was not restored by TRAIL deficiency. Recently, TRAIL has been shown to possess antiviral function against dengue virus (39); however, we observed no defects by TRAIL^−/− mice in the ability to control i.n. VV-WR infection (data not shown). Collectively, the results imply that the impaired recall response by helpless memory cells in VV-WR infection is not due to increased cell death and is independent of TRAIL.

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FIGURE 6.

The defective recall response by helpless memory cells is independent of TRAIL. A, VV-specific memory CD8⁺ T cells from undepleted, CD4-depleted/R-IgG-treated, or CD4-depleted/anti-CD40 treated mice were adoptively transferred into naive recipients and challenged as shown in Fig. 5C. Five days postchallenge, donor-derived VV-specific CD8⁺ T cells were stained with annexin V or by intracellular Bcl-2. Representative plots from two independent experiments with six mice per group are shown. Left, Representative plot (gated on donor cells) showing gating on tetramer⁺ cells. Right, Gated on CD45.2⁺CD8⁺tetramer⁺ cells. B, The recall response of VV-specific memory CD8⁺ T cells from undepleted B6, CD4-depleted B6, or CD4-depleted TRAIL^−/− mice was analyzed by adoptive transfer and challenge as shown in Fig. 2D. Numbers indicate average fold expansion of VV-specific memory cells for each group. Each circle represents an individual recipient. Representative data from two independent experiments using four donor mice per group and four to six recipient mice per group are shown.

Excessive up-regulation of PD-1 on helpless memory cells impairs the recall response

Data suggesting that the defective recall response of helpless memory CD8⁺ T cells is independent of TRAIL-mediated cell death prompted us to investigate other pathways that may mediate the helpless defect. We hypothesized that during recall, helpless memory cells express higher levels of receptors that inhibit T cell proliferation, reducing the magnitude of the recall response. We explored two immunoregulatory receptors in the B7-CD28 superfamily, CTLA-4 and PD-1, which are known for their potent inhibitory role in the T cell response. VV-specific memory CD8⁺ T cells from undepleted, CD4-depleted/R-IgG-treated, or CD4-depleted/anti-CD40-treated mice were challenged upon transfer, and 5 days later they were assessed for the expression of these molecules. The expression of CTLA-4 during recall did not correlate with the ability of the memory cells to mount a recall response (data not shown), indicating that the molecule does not account for the difference in proliferative capacity between helped and helpless memory cells. Next, we examined the expression of PD-1 on memory CD8⁺ T cells during the recall response. The expression of PD-1, both in terms of percentage of VV-specific CD8⁺ T cells expressing the receptor and the MFI, was significantly increased on memory CD8⁺ T cells that did not receive CD4 help (Fig. 7⇓, A–C). Furthermore, restoring the recall response in CD4-depleted mice by administering agonistic anti-CD40 mAb resulted in the down-regulation of PD-1 expression during the recall response. Thus, expression of PD-1 during the recall response correlated with CD4 help and CD40 signaling, as well as the proliferative capacity of helped vs helpless memory cells during recall. PD-1 expression was not observed on resting memory cells before recall in all groups (Fig. 7⇓D).

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FIGURE 7.

Helpless memory cells express excess levels of PD-1 during recall compared with helped memory cells. VV-specific memory CD8⁺ T cells from undepleted, CD4-depleted/R-IgG-treated, or CD4-depleted/anti-CD40 treated mice were adoptively transferred into naive recipients and challenged as shown in Fig. 5C. Five days postchallenge, donor-derived VV-specific CD8⁺ T cells were stained for PD-1 expression. A, Representative plots gated on CD45.2⁺CD8⁺tetramer⁺ cells are shown, and numbers indicate the average percentage of VV-specific CD8⁺ T cells expressing PD-1. B, Graphed presentation of A. C, MFI of PD-1 expression by donor-derived VV-specific CD8⁺ T cells. Representative data from two independent experiments with six recipients per group are shown. D, PD-1 expression on resting VV-specific helped and helpless memory CD8⁺ T cells at 49 days postinfection. Representative data from two independent experiments with three to four mice per group are shown. Plots are gated on CD8⁺tetramer⁺ cells. Error bars indicate SEM. ∗, p > 0.05 and ∗∗, p > 0.01.

Because the expression of PD-1 was higher on helpless memory cells during recall compared with helped cells, we hypothesized that the level of PD-1 expression may play a role in blunted recall responses in the absence of CD4 help. To investigate the functional significance of PD-1 expression, we inhibited PD-1 on helpless memory cells during the recall response by Ab blockade. Treatment with anti-PD-1 during recall significantly enhanced the recall response of helpless memory cells, to the levels of helped memory cells (Fig. 8⇓A). Interestingly, treatment with anti-PD-1 had no effect on annexin V staining (Fig. 8⇓B) or Bcl-2 expression (data not shown) on the virus-specific CD8⁺ T cells, implying that PD-1 inhibits the recall response without affecting cell survival. PD-1 blockade also enhanced recall responses of helped memory cells (Fig. 8⇓C), which is in line with our observation of PD-1 up-regulation on hel