Multiple Redundant Effector Mechanisms of CD8⁺ T Cells Protect against Influenza Infection

Adoptive transfer of CD8⁺ effectors brings about a reduction in viral load and lung pathology

We have previously shown (15–17) that all three subsets of cells can protect naive mice from otherwise lethal challenge, but our previous studies had measured only weight changes and survival. We show in this study that injection of Tc1 or Tc17 effectors reduces initial viral load (Supplemental Fig. 1A), prevents damage as shown by reduction in leakage of albumin (Supplemental Fig. 1B) and lactate dehydrogenase (Supplemental Fig. 1C) into the lung lavage, and alleviates respiratory distress as shown by changes in minute volume (Supplemental Fig. 1D) and respiratory rate (Supplemental Fig. 1E).

In vivo–generated polyclonal CD8⁺ effectors also provide protection

It is not possible to prepare well-polarized CD8 effectors in vivo, but polyclonal CD8 effectors were isolated from mice challenged with 0.5 LD PR8 7 d postinfection, and graded numbers were injected into naive recipients that were then challenged with 3 LD PR8. As few as 4 × 10⁶ effectors reduced weight loss and increased survival, establishing that protection was not unique to the use of TcR transgenic T cells (data not shown).

CD8⁺ effectors that lack perforin-mediated cytotoxic activity are still protective

The prevailing belief is that CD8⁺ T cells protect against viral infection by killing virally infected cells. We have previously shown that Tc1 and Tc17 effectors are equally protective even though Tc1 are lytic in vitro, whereas Tc17 are not (16). We showed also that protection by Tc1 was diminished in effectors made from perforin^−/− mice, whereas protection by Tc17 was not (16). Although Tc17 effectors lacked FasL or TRAIL expression in vitro (Supplemental Fig. 2), it remained possible that Tc17 develop in vivo lytic activity dependent on FasL or TRAIL expression on injection back into the animal. To examine this possibility, we injected Tc17 effectors from OT-1 mice into uninfected naive recipients and looked for killing of SIINFEKL-labeled targets at 24 h in an in vivo cytolysis assay.

When Tc17 effector cells are injected into normal mice, they do show some killing activity at 24 h after injection, but less activity than Tc1 (Fig. 1A). The Tc17-mediated killing, however, is FasL dependent because Fas-negative targets from lpr mice are not killed (Fig. 1B). Eventually, however, Tc17 cytolytic activity develops and becomes quantitatively equivalent to that of Tc1 by day 5 (Fig. 1C), and many of the injected cells become double producers of IFN-γ and IL-17 (data not shown).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 1.

Changes in the cytotoxic activity of adoptively transferred CD8 effector T cells. A total of 4 × 10⁶ in vitro–generated Tc1 or Tc17 OT-1 effectors were injected into groups of five naive B6 recipients. Twenty-four hours later, CFSE high-concentration-labeled, SIINFEKL-pulsed target cells and CFSE low-concentration-labeled control cells were injected. Twenty-four hours later, mice were sacrificed and the ratio of high to low CFSE-labeled cells was determined by flow cytometry to measure the level of in vivo killing by the OT-1 effectors. (A) Tc17 and Tc1 at 24 h. In another assay (B), target cells were made from WT or Fas-deficient lpr mice to determine whether the killing was FasL dependent. **p < 0.01, ****p < 0.0001. (C) The ex vivo killing assay was performed with Tc17 or Tc1 purified from mice previously injected with Tc1 or Tc17 effectors, then infected with PR8-OVA_I. Five days postinfection, effectors were isolated and injected into naive B6 mice. One day later, CFSE-labeled target cells were then transferred to 5-d postinfection effector-transferred mice and killing was measured [as labeled in (A)]. (D and E) A total of 8 × 10⁶ in vitro–generated Tc17 or Tc1 effectors from perforin^−/− or WT mice were injected into WT (D) or lpr (E) mice, challenged with 2 LD₅₀ PR8-OVA_I, and weight changes and survival followed for 11 d. Similar results were seen in two or more experiments for each panel.

To determine whether FasL-mediated killing plays a role for Tc17-mediated protection, we injected Tc1 or Tc17 effectors from WT or perforin-deficient mice into WT or lpr recipients to determine whether they could still protect in the absence of both perforin and FasL-mediated killing. WT Tc17, WT Tc1, and perforin-deficient Tc17 were fully protective in WT recipients (Fig. 1D), but mice that received Tc1 from perforin-deficient mice lost more weight (Fig. 1D) and two of four died. This differential was more marked when the same cells were transferred into lpr recipients where Tc1 cells from perforin-deficient mice provided no protection, whereas Tc17 WT or perforin-deficient effectors were still protective (Fig. 1E). We conclude that although perforin-mediated lysis is important in the protection mediated by Tc1 and Tc2 cells, neither perforin nor FasL-mediated killing play a significant role in the protection mediated by Tc17.

In further experiments, we attempted to determine whether Tc1 or Tc17 protection is mediated by a TRAIL-dependent mechanism using Tc1 and Tc17 effectors prepared from CD8⁺ T cells from OT-1.TRAIL^−/− mice. Tc17 effectors from TRAIL-deficient mice were still able to protect (data not shown), but a high proportion of the CD8⁺ cells in the naive OT-1.TRAIL^−/− mice were CD44^high, and it was not possible to make preparations of Tc17 effectors from these mice with anything more than a very low percentage of IL-17–secreting cells. We were thus unable to completely exclude the possibility that Tc17 effectors protect by a TRAIL-mediated lytic mechanism, as suggested by Brincks and colleagues (19). We turned next to other correlates associated with the protection mediated by the injection of CD8⁺ effectors to determine whether any were essential for protection.

Adoptive transfer of CD8 effectors enhances recruitment of host cells

CD4⁺ and CD8⁺ T cells, B cells, neutrophils, NK cells, and macrophages are all recruited into the lungs of influenza-infected mice (20, 21). The injection of already functional CD8⁺ effectors from OT-1 mice accelerates the recruitment of most of these cell types after exposure to PR8-OVA_I. Differential effects are seen with effectors of different subsets; thus, Tc2, for example, recruit greater numbers of eosinophils than Tc1 (17), and Tc17 recruit more B cells (see later). We had also previously shown that the injection of Tc17 effectors led to a greater accumulation of neutrophils than Tc1 after viral challenge (16). In further experiments, we found that after the adoptive transfer of either Tc1 or Tc17 effectors, Tc17 effectors recruited greater numbers of cells to the lung than Tc1 recipient or control mice (Fig. 2A), and that Tc17 cells entered the lung more rapidly and in greater numbers than Tc1 cells (Fig. 2B). Tc17 effectors were able to recruit host CD4⁺ (Fig. 2C) and CD8⁺ T cells (Fig. 2D) more effectively than Tc1 effectors. However, host CD4⁺ T cells rapidly declined in the recipients of CD8⁺ effectors after day 10, whereas they continued to increase in the untreated controls, and the net effect of the transfer was actually to decrease recruitment of host T cells at later time points. There was, however, a striking increase in the number of B cells (Fig. 2E) after adoptive transfer of Tc17, which persisted through day 20; clusters of B cells were seen in the lungs of mice 8 d after transfer of Tc17 cells (Supplemental Fig. 3). We concluded that the cytokines and chemokines released after the transfer of Tc17 and, to a lesser extent, Tc1 led to an early enhancement of recruitment of many cell types, including neutrophils, NK cells, macrophages, and B cells. This correlated with the control of the viral load and was followed by a decline in the numbers of recruited T cells and, to a lesser extent, B cells in the treated mice, whereas the numbers continued to increase in the untreated controls.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 2.

Total cells (A), donor CD8⁺ (B), CD4⁺ (C), host CD8⁺ (D), B cells (E), and CD19⁺ B cells are recruited to the lung after adoptive transfer of CD8 effector T cells and viral challenge. Groups of five B6 mice were injected with 8 × 10⁶ in vitro–generated Tc1 (□) or Tc17 Thy-1.1.OT-1 effectors (▪) or left uninjected (△) and challenged 1 d later with 0.2 LD₅₀ PR8-OVA_I. Mice were sacrificed at the times indicated, and cell suspensions were prepared from the lungs and stained with fluorescently labeled Ab to prosurfactant C to determine the numbers of donor (Thy-1.1⁺) and host cells recruited into the lungs. Similar results were seen in two experiments.

Protection is accompanied by an accelerated regeneration of type II epithelial cells in the lung

Mice were protected from lethal challenge by the transfer of Tc1 or Tc17 effectors. Mice were sacrificed at days 4, 6, and 8 after challenge, and the lungs were analyzed by examination of stained lung sections and by flow cytometry of lung cell suspensions. Lung sections were stained with Ab to prosurfactant protein C (as a marker for type II epithelial cells) and the numbers of positive cells counted by field in mice receiving Tc1 or Tc17 effectors or left untreated. Lung cell suspensions were also prepared and stained and analyzed by flow cytometry. It can be seen (Fig. 3) that there was a highly significant accelerated increase in the number of stained cells both by histology and by FACS, suggesting more rapid recovery of lung epithelium in treated mice, but whether this represents the cause or effect could not be deduced. Tc17 effectors were more effective than Tc1.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 3.

Accelerated regeneration of type II epithelial cells in protected mice. (A) Groups of five B6 mice were injected with 8 × 10⁶ in vitro–generated Tc1 (▲) or Tc17 Thy-1.1.OT-1 effectors (▪) or left uninjected (●) and challenged 1 d later with 0.2 LD₅₀ PR8-OVA_I. Mice were sacrificed at the times indicated, and lung sections were prepared and stained with fluorescently labeled Ab to prosurfactant protein C as a marker for type II epithelial cells to determine the numbers of positive cells per field. For the differences between Tc17 versus no transfer at days 6 and 8, p < 0.001, for Tc1 versus no transfer p < 0.01, and for Tc17 versus Tc1, p < 0.01. (B) Cell suspensions from the lungs of similarly treated mice were prepared from mice sacrificed at day 8 and analyzed with the same fluorescent-labeled Ab by flow cytometry. Tc17 versus none, Tc1 versus none, and uninfected versus none, all *p < 0.05.

The earlier findings suggested a considerable number of candidate mechanisms for protection, and we sought to confirm their role by showing a reduction or abrogation of protection in their absence. Our next step was to determine whether any of the identified potential mechanisms were essential for protection.

Protection is still seen when host neutrophils are depleted

Neutrophils are generally thought to exacerbate immunopathology in viral infections, but we had seen an early spike in neutrophil numbers in studies of heterosubtypic protection (data not shown) that was accompanied by an early reduction of the viral load. We speculated that an early, moderate influx of neutrophils might be beneficial to the outcome of the infection, whereas the damage done by larger numbers at later times was a correlate of the failure to control infection.

Two groups of naive B6 mice were injected with 8 × 10⁶ Tc17 effectors at day zero and infected with 2 LD₅₀ PR8-OVA_I to test the role of neutrophils in infection. One group of five mice was injected with 200 μg of the mAb, 1A8, to deplete the Ly6G-high neutrophils on days −1, +1, and +3, whereas the second group was injected with an equivalent amount of isotype control Ig. Two additional groups of mice were run in parallel, receiving Tc1 rather than Tc17. Additional mice from each protocol were sacrificed to check for neutrophil depletion as judged by staining with fluorescently labeled anti–GR-1 (RB6) Ab. A fifth group of mice received no CD8⁺ effectors and no Ab, but were challenged with virus. Weight loss and survival were followed for 28 d. No effects were observed on either weight changes or survival (Fig. 4A) after effective neutrophil depletion (Fig. 4B). We concluded that neutrophil recruitment does not play an essential role in protection.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 4.

Depletion of host neutrophils or NK cells does not diminish protection. A total of 8 × 10⁶ in vitro–generated Tc1 or Tc17 OT-1 effectors were injected into groups of five naive B6 recipients. Mice were challenged with 2 LD₅₀ PR8-OVA_I. (A) Mice were depleted of neutrophils or received isotype control Ig, and weight changes and survival were tracked for 30 d. The effectiveness of the neutrophil depletion is shown in (B). (C) Mice were depleted of NK cells or received isotype control Ig, and weight changes and survival were tracked for 13 d. The effectiveness of the NK cell depletion is shown in (D). The details of all of the procedures are as described in Materials and Methods. Similar results were seen in two experiments.

Protection is still seen when host NK cells are depleted

A similar experiment was carried out to determine whether NK cell depletion, using 200 μg NK-1.1 Ab, injected on days −1, +2, and +5, would affect the level of protection. Again, there was no effect on either weight loss or survival (Fig. 4C) after effective NK cell depletion (Fig. 4D). We concluded that NK cell recruitment does not play an essential role in protection.

Protection is still seen when host T cells are absent

Although adoptive transfer actually decreased the recruitment of host T cells at later time points, we considered it possible that they might still play some role in the early clearance of virus. We therefore determined the ability of Tc1 and Tc17 effectors to protect mice lacking both CD4⁺ and CD8⁺ T cells. For this we used TCRβ/TCRδ-deficient recipient mice. Uninjected β/δ^−/− mice lost weight rapidly, and all died by day 10 when challenged with 3 LD₅₀ PR8-OVA_I (Fig. 5A). β/δ^−/− mice injected with either 8 × 10⁶ Tc17 or Tc1 effectors at day −1 started to regain weight by day 5 and were fully recovered by day 10. Individual mice began to lose weight again around day 30 and eventually died (data not shown), possibly after development of viral escape mutants, although this was not analyzed. We concluded that neither recruited host CD4⁺ nor CD8⁺ T cells are crucial for early protection.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 5.

Tc1 and Tc17 protect TCRβ/TCRδ^−/− recipient mice or RAG-2^−/− mice from viral challenge. A total of 8 × 10⁶ in vitro–generated Tc1 (□) or Tc17 (▪) OT-1 effectors were injected into groups of five naive recipients. Recipients were either B6.TCRβ/TCRδ^−/− (A) or B6.RAG-2^−/− mice (B). In (A) and (B), a control group of recipient mice was left uninjected (△). Mice were challenged with ∼2 LD₅₀ of PR8-OVA_I for TCRβ/TCRδ^−/− and RAG-2^−/− mice, respectively, and the weight changes followed for 28 d. Similar results were seen in an earlier experiment in which the mice were challenged with only 0.2 LD₅₀.

Protection is still seen when host T cells and B cells are absent

Finally, we examined whether protection could be seen in the absence of both αβ and γδ T cells and B cells. A total of 8 × 10⁶ Tc1 or Tc17 effectors were injected into RAG-2^−/− host, which were challenged with 3 LD₅₀ PR8-OVA_I. Yet again, the injected effectors were able to reverse weight loss and protect the mice for at least 15 d as shown in Fig. 5B. As with the β/δ-deficient recipient mice, individual mice started to die at later time points (data not shown), but, again, it was clear that none of absent cell types was required to generate the initial protection.

Protection is still seen when multiple cell lineages are absent or depleted and donors are perforin deficient

In a further attempt to show clusters of mechanisms that, collectively, were essential for protection, we engineered multiple deficiencies into a single protocol. Tc1 (Fig. 6A, 6B) or Tc17 (Fig. 6C, 6D) effectors were prepared from CD8⁺ T cells from either WT or perforin-deficient OT-1 mice, and these were injected into WT (Fig. 6A, 6C) or RAG-2^−/− (Fig. 6B, 6D) recipients followed by infection with 3 LD₅₀ PR8-OVA_I. In addition, a second set of recipients was injected with the same sets of Tc17 cells but was also subjected to the regimen previously used to deplete neutrophils (Fig. 6C, 6D). Thus, in the most compromised protocol, the recipient mice lacked four of the postulated protective mechanisms, CD4⁺ and CD8⁺ T cells, B cells, and neutrophils; in addition, the donor cells were deprived of perforin-mediated killing. Under these conditions, the Tc17 perforin-deficient effectors were still able to protect RAG-2–deficient recipients from lethal challenge, even when neutrophils were depleted (Fig. 6C, 6D). Tc1 effectors from perforin-deficient donors, however, were less protective than Tc1 effectors from WT mice in RAG-2^−/− hosts, again exposing a difference in the mechanism of protection by Tc17 and Tc1 effectors.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 6.

Perforin-deficient Tc17 protect neutrophil-depleted RAG-2^−/− recipients from viral challenge. The design of the experiment was similar in design to that in Fig. 4B, except that the CD8 effectors were generated with cells from WT or perforin-deficient mice and the recipients were left untreated or were injected as in Fig. 3A to deplete neutrophils. All mice were challenged with 2 LD₅₀ PR8-OVA_I. Similar results were seen in two experiments. (A) and (B) indicate the kinetics of weight change for WT mice (A) that were injected with Tc1 WT or perforin-deficient effectors and for RAG-2^−/− mice (B) that were injected with Tc1 WT or perforin-deficient effectors. (C) and (D) indicate WT mice that were injected with Tc17 WT or perforin-deficient effectors (C), and for RAG-2^−/− mice that were injected with Tc17 wt or perforin-deficient effectors (D). In addition, in (C) and (D), the recipient mice were either neutrophil depleted or left untreated. Similar results were seen in three experiments.

We were somewhat surprised at the robustness of the protective effect thus revealed, and next attempted to determine which parts of the protective effect were Ag specific and which were Ag nonspecific. We carried out experiments in two models: in the first, to determine how long the donor cells needed to be present in the recipient after transfer to retain protection; and in the second, to determine whether protection against the specific virus provided any bystander protection against a virus lacking the SIINFEKL insert, given at the same time.

Donor cells can be depleted at later times after transfer without loss of protection

We injected 8 × 10⁶ Tc1 or Tc17 effectors from B6.OT-1.Thy-1.1 mice into WT naive B6.Thy-1.2 recipients on day zero and then challenged with 1 LD₅₀ PR8-OVA_I. Mice in one group of five mice were left without further manipulation and were protected as before. Mice in a second group were injected with anti–Thy-1.1 to eliminate the donor cells at day 3, mice in a third group were injected at day 5, and mice in a fourth group at day 8. Control, no transfer mice (the same curve for the same mice is shown in the left panels of Fig. 7A, 7B) lost weight, and some died at days 7 and 8, but the remainder began to regain weight as shown in the survival plots (survival panels in Fig. 7A, 7B). Mice injected with Tc1 effector T cells showed little difference in weight change (Fig. 7A, left panel), but most survived. If the Tc1 were depleted at day 3, the recipients lost more weight (Fig. 7A, middle panel) and fewer survived (Fig. 7A, right panel). Depletion at days 5 and 8 had little effect.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 7.

Tc17 donor cells still protect against viral challenge when depleted at day 5 or 8, but not at day 3. A total of 8 × 10⁶ in vitro–generated Tc1 (A) or Tc17 (B). OT-1 effectors were injected into groups of five naive WT B6 recipients and challenged 1 d later with ∼1 LD₅₀ PR8-OVA_I. Donor T cells were from OT-1.Thy-1.1 mice and were depleted by the injection of anti–Thy-1.1 at day 3, 5, or 8. Weight changes and survival were followed for 15 d. Data displayed in (A) and (B) all come from the same experiment, but for ease of display, the weight change curves are separated into two parts. The weight changes for untreated mice (no transfer) and the isotype control for the CD8 effector treated mice are shown in the left panels of (A) and (B). The effects of depletion at 3, 5, and 8 d are shown in the middle panels. The corresponding survival curves for (A) and (B) are shown in the right panels. (C) Representative example of the efficacy of donor cell depletion using anti–Thy-1.1 injection. Similar results were seen in two experiments.

A similar pattern was seen for the Tc17 half of the experiment (Fig. 7B), except that Tc17 effectors were more protective (Fig. 7B, left panel). Depletion of Tc17 effectors at day 3 resulted in slightly more weight loss, whereas depletion at days 5 and 8 was without effect (Fig. 7B, center panel). All Tc17-treated mice survived whenever they were depleted (Fig. 7B, right panel).

The efficacy of the donor cell depletion by injection of anti–Thy-1.1 is illustrated in Fig. 7C, which shows that the percentage of Thy-1.2⁻ CD8⁺ donor cells was reduced from 22.5 to 0.52.

Note that all of the weight change data presented in Fig. 7A and 7B are from a single experiment but are separated into different panels so that the weight loss curves may be distinguished. This pattern of results was seen in each of two experiments and suggests that an initial period that requires the continued presence of the donor cells is followed by, at day 5, a period in which the original injected cells are no longer required. This implies that the CD8 effectors had set in motion a train of protective events that no longer required their presence. The requirement for the persistence of Tc1 effectors would appear to be more prolonged for Tc17-mediated protection.

The OT-1 response to the OVA-bearing virus (PR8-OVA_I) provides bystander protection against the virus lacking the OVA epitope (A/PR8)

Mice were injected with Tc1 (Fig. 8A) or Tc17 (Fig. 8B) effectors generated from OT-1 mice and were challenged with ∼1 LD₅₀ of the “specific” virus, PR8-OVA_I, or a lethal dose of the nonspecific virus, 4 LD₅₀ PR8, or both.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 8.

Tc17 donor cells provide bystander protection against viral challenge. A total of 8 × 10⁶ in vitro–generated Tc1 (A) or Tc17 OT-1 (B) effectors were each injected into three groups of mice. One was challenged PR8, a second with PR8-OVA_I, and the third with both viruses (n = 4). (C) The third group of mice was injected with Tc1 effectors and 100 μg LPS-free OVA i.n. instead of PR8-OVA_I (n = 5). Weight changes and survival were followed for 15–30 d.

All the mice receiving Tc1 effectors from OVA peptide-specific OT-1 mice died when challenged with PR8 alone, whereas one of four survived when challenged with both viruses and regained weight (Fig. 8A). All mice challenged with 1 LD₅₀ PR8-OVA_I lost less weight, recovered, and survived. The difference was more marked when mice received Tc17 effectors (Fig. 8B). Three of four mice challenged with PR8 lost weight rapidly and died. Mice challenged with both viruses also lost weight as rapidly as those challenged with PR8 alone but started to recover at day 10, and three of four survived, suggesting that the addition of the specific virus led to protection against the nonspecific virus. Only one of four mice survived PR8 alone. A similar pattern of reduced weight loss when both viruses were given was seen in a second experiment in which a lower challenge dose of PR8 was used. In a further experiment, we used LPS-free OVA instead of PR8-OVA_I and again observed a bystander effect with protection against a lethal challenge with PR8 (Fig. 8C). This eliminates the possibility that the apparent bystander effect was really because SIINFEKL-specific T cells killed doubly infected cells and the possibility that the two viruses in some way compete with one another. There was a small reduction in viral load at day 3 in mice given PR8 plus OVA compared with PR8 alone, but it was not sustained and the titer had rebounded by day 7 (Supplemental Fig. 4).

We conclude that, together, the effector depletion and the bystander protection experiments support a model in which an Ag-specific step leads to a subsequent protective mechanism that is no longer Ag specific.

The transfer of CD8⁺ rested effectors leads to activation of an early host innate response

We have previously shown that the adoptive transfer of memory CD4⁺ cells can bring about an induction of a number of innate cytokines and chemokines early in the response to influenza infection (22), and that this was correlated with a 10-fold reduction in viral titer and an increase in survival. CD8⁺ effectors express many of the same effector mechanisms expressed in CD4⁺, and it seemed possible that they too might elicit early innate responses.

We found, in this study, that the adoptive transfer of rested CD8⁺ effectors from the HA-specific TcR transgenic clone 4 BALB/c mice were also able to induce this same early response (Fig. 9). In this experiment, we used effectors that were rested 3 d before transfer. Such cells adopt a memory phenotype (23) but become reactivated to effectors on transfer to infected mice (24). The rested CD8⁺ effectors were transferred into naive BALB/c recipients, which were then challenged with 2 LD₅₀ PR8. The mice were sacrificed at days 1, 2, or 3, and the supernatant from the lung homogenates assayed for a panel of cytokines and chemokines using Luminex. Mice injected with memory CD4 T cells show enhanced survival (25) after viral challenge, and memory CD8 are also effective (data not shown). The factors indicated were induced by day 2, and the level of induction was comparable with that seen with CD4⁺ cell transfer (22).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 9.

CD8 rested effector responses in the lung acutely enhance a broad panel of cytokine and chemokines. Rested effectors were prepared by 4-d in vitro stimulation of CD8 cells from clone 4 mice bearing the TCRs for the IYSTVASSL peptide from the HA of PR8. The effectors were then washed and cultured in the absence of cytokines for a further 3 d. A total of 5 × 10⁶ naive or CD8 rested effectors were injected into naive BALB/c mice, then challenged with 2 LD₅₀ PR8. Mice were sacrificed on day 1, 2, or 3, and the supernatants of lung homogenates assayed for the cytokines and chemokines indicated by Luminex. Similar results were seen in two experiments.