As previously reported, the CL-4 Tg CD8+
T cells express a TCR that recognizes an epitope (residues 533–541) within the trans-membrane domain of the A/PR/8 influenza virus HA primary translation product in association with H-2Kd
). Stimulation of this T cell population with syngeneic, irradiated splenocyte APCs infected with the A/PR/8 virus triggers robust proliferation that is exemplified by an increase in cell numbers, as determined by trypan blue counting (). Stimulation of the CL-4 Tg T cells with splenocyte APCs infected with the A/Japan influenza virus, which contains a variant epitope (residues 529–537) that differs by two amino acids from the PR/8 epitope, triggers a similar proliferative response through day 3 poststimulation (). On day 4 of culture, however, there was a dramatic reduction in the number of CL-4 T cells responding to Japan virus stimulation, with most of the cell loss occurring between days 3 and 4 (). We considered the possibility that the differential response of the CL-4 T cells could be due to intrinsic properties of the different influenza viruses rather than to specific recognition of the viral epitopes. To address this, we stimulated CD8+
CL-4 T cells with splenocytes pulsed with synthetic PR8 HA533–541
or Japan HA529–537
peptide and examined cell counts by trypan blue exclusion at various times poststimulation (). We found that stimulation with either the PR8 or Japan peptide induced an increase in cell numbers through day 3 poststimulation; however, there was a significant decrease in cell numbers in response to the Japan variant epitope between days 3 and 4, which was consistent with the results obtained when virus-infected splenocytes were used to stimulate the T cells (). These data suggest that the differential response of the CL-4 T cells was due to specific recognition of the agonist PR8 and variant Japan viral epitopes, and that Japan epitope stimulation results in the death of the responding CL-4 T cells. It should be noted that the Japan HA529–537
epitope efficiently binds to H-2Kd
and serves as an agonist to stimulate a vigorous in vivo primary anti-viral CD8+
effector T cell response (18
FIGURE 1 Stimulation with a variant viral epitope induces enhanced apoptosis in CD8+ CL-4 T cells. CL-4 T cells were stimulated in vitro with A/PR/8/34 or A/Japan/305/57 influenza virus-infected splenocytes (A) or with 10−5 M (B) or 10−6 M (C, (more ...)
We next assessed the viability of the CD8+ CL-4 T cells stimulated with the agonist or variant epitopes by flow cytometric detection of their forward and side light-scattering properties. To first establish the scatter profile of live and dead cell populations, fluorescently conjugated Annexin V was used to label apoptotic cells. As shown in , all of the T cells within a traditional lymphocyte or live cell gate were not efficiently bound by annexin V, confirming that they were live lymphocytes. In contrast, cells with reduced forward scatter (smaller size) and increased side scatter (higher granularity) bound annexin V well. We termed this a dead gate (). Using this defined live and dead cell gating strategy, we then examined the survival of the CD8+ CL-4 T cells after stimulation with either the PR8 HA533–541 or Japan HA529–537 peptide (). At early times poststimulation (days 1–2), T cells responding to either the agonist or variant peptide were present within the live lymphocyte gate. However, obvious differences in the culture were observed at later time points. Although the majority of agonist epitope-stimulated T cells remained in the live lymphocyte gate until day 5 poststimulation, >90% of variant epitope-stimulated T cells accumulated in the dead cell gate by day 4 (). These initial observations suggest that stimulation with the variant viral epitope induces accelerated and enhanced apoptosis of CD8+ CL-4 T cells.
Because these initial in vitro observations could be related to the nonphysiologic stimulation of CL-4 T cells with high doses of synthetic peptide or virus, we next sought to determine whether CL-4 T cells would undergo extensive cell death in response to natural virus infection in vivo. To this end, we adoptively transferred CD8-purified, CFSE-labeled Thy1.2+
CL-4 T cells into naive Thy1.1+
recipient mice, allowing early detection and quantitation of donor CD8+
T cells in vivo based on Thy1.2 iso-form expression. One day after cell transfer, recipient mice were infected intranasally with a sublethal dose of PR/8 or Japan influenza virus. The total numbers of transferred CD8+
Tg T cells in the draining mediastinal lymph nodes (MLNs), lung, and spleen were determined on successive days postinfection (). As previously reported, donor T cells were detectable throughout the lymphoid tissue of recipient mice, including the lung-draining MLNs, peripheral nondraining lymph nodes, and spleen after transfer (data not shown) (16
). The onset of donor T cell proliferation in the MLNs occurred at day 3 postinfection and continued through days 4–5. Although both the Japan and PR8 viruses triggered similar CL-4 T cell proliferative responses in the MLNs at day 4 postinfection, there was a dramatic reduction in the total number of CL-4 T cells in the MLN 1 d later in Japan virus-infected compared with PR8 virus-infected mice (). We reasoned that the loss of CL-4 T cells in the MLNs of Japan-infected mice could result from the migration of these cells to other organs, such as the lung or spleen; however, the low numbers of Tg T cells present in these organs at day 5 postinfection could not account for the significant loss of cells from the MLN between days 4 and 5 postinfection (). These results demonstrating a selective loss of CD8+
CL-4 T cells responding to natural Japan virus infection in vivo suggest that the enhanced apoptosis we observed in vitro is a physiologic response of the these cells to stimulation with the variant Japan epitope and not an artifact of high-dose peptide stimulation.
FIGURE 2 Selective loss of CD8+ CL-4 T cells in response to the altered Japan epitope following natural virus infection in vivo. Purified CD8+ Thy1.2+ splenic CL-4 T cells were labeled with CFSE and injected i.v. into Thy1.1+ recipient mice that were infected (more ...)
Studies have suggested that some APLs may alter the outcome of T cell priming by providing a weak or suboptimal TCR signal. Therefore, we considered the possibility that the marked difference in viability of the CL-4 T cells stimulated with the agonist and variant viral peptides might result simply from the Japan peptide triggering suboptimal TCR stimulation. If this were the case, we hypothesized that stimulation of the CL-4 T cells with lower doses of the agonist PR8 peptide should mimic the phenotype of variant epitope-stimulated cells. To address this, we examined the response of CD8+ CL-4 T cells after stimulation with splenocytes pulsed with a range of PR8 HA533–541 or Japan HA529–537 peptide concentrations (10−9–10−5 M; ).
FIGURE 3 Peptide dose response of CD8+ CL-4 T cells. A, CL-4 T cells were stimulated in vitro with the indicated concentrations of PR/8 HA533–541 or Japan HA529–537 peptide-pulsed splenocytes. At the indicated times poststimulation, viable cell (more ...)
Although lower doses (10−9–10−8M) of the variant peptide appear to be less efficient at triggering the CL-4 T cells than the agonist peptide at these same concentrations, we were unable either to reverse the accelerated apoptosis observed following stimulation with the variant epitope by increasing variant peptide concentrations used for stimulation or to accelerate the apoptosis of naive CL-4 T cells stimulated with the agonist epitope at low peptide concentrations (). At peptide concentrations <10−9 M, the variant Japan epitope was minimally stimulatory, whereas the agonist PR8 epitope decreased in stimulation efficiency (i.e., the fraction of CL-4 T cells responding at progressively lower peptide concentrations) (data not shown).
As discussed below, CL-4 T cells stimulated with the agonist PR8 epitope differentiated into effector T cells. This provided an opportunity to determine whether these activated CL-4 effector cells differed in their functional avidity for the agonist and variant epitopes. To this end, we evaluated the peptide dose-dependent of IFN-γ production by the activated CL-4 effector cells in the intracellular cytokine assay. As demonstrates, whereas both ligands elicited comparable IFN-γ production at higher peptide doses, the efficiency of recognition of the variant Japan epitope decreased significantly in the triggering of IFN-γ production at peptide concentrations below 10−9 M. This finding suggested that the CL-4 T cells differed in functional avidity for the agonist and variant epitopes.
IL-2 has been well recognized to have both pro-proliferative and anti-apoptotic effects on responding T cells (as well as the capacity to prevent or reverse APL-induced anergy) following engagement of the high affinity IL-2 receptor. We asked whether accelerated apoptosis induced by stimulation of naive CL-4 T cells with variant epitopes could be reversed by stimulation of the T cells with the variant epitope in medium supplemented with exogenous IL-2. As demonstrates, IL-2 supplementation of cultures at 40 U/ml had no effect on T cell viability or the tempo of induction of apoptosis. Similar results were obtained over a range of IL-2 concentrations (10–100 U/ml, data not shown).
The proliferation of naive T cells responding to specific Ag is thought to be linked to their differentiation and acquisition of effector activity. To characterize the response of CD8+ CL-4 T cells to agonist and variant epitope stimulation in vitro, we first analyzed their proliferative capacity by CFSE dilution. The agonist and variant peptides triggered a similar onset and rate of cell division in CD8+ CL-4 T cells (). In both cases, the T cells began proliferating at day 2 and had undergone 6 divisions by 3 d poststimulation. However, there was a significant loss of divided (CFSE-low) CL-4 T cells at days 3–4 in cultures stimulated with the variant peptide, whereas cultures stimulated with the agonist peptide continued to accumulate cells (). Similar data were obtained using virus-infected splenocytes as stimulators (). These results are consistent with the trypan blue cell count data in and suggest that although the initial proliferation of CD8+ CL-4 T cells responding to the altered influenza epitope is normal, the survival of these cells is markedly impaired.
FIGURE 4 CD8+ CL-4 T cells proliferate comparably in response to agonist and variant epitope stimulation. CFSE-labeled CD8+ CL-4 T cells were stimulated in vitro with 10−6 M PR/8 HA533–541 or Japan HA529–537 peptide-pulsed splenocytes ( (more ...)
To further evaluate the effects of agonist and variant epitope stimulation on CL-4 T cells, the cells were examined for their expression of proteins whose expression is tied to the activation/differentiation state of the cells following antigenic stimulation. As shown in , CD8+
CL-4 T cells from both agonist and variant epitope-stimulated cultures upregulated the critical activation markers, CD25 and CD69, to a similar extent at days 1–2 post-stimulation. Likewise, agonist and variant peptide-stimulated CL-4 T cells were able to rapidly produce IFN-γ, TNF-α, and IL-2 early after stimulation, suggesting that these cells had become mature effectors (). Cytotoxicity is another hallmark feature of mature effector CD8+
T cell differentiation, with cytolysis linked to the expression of lytic granule-associated molecules, such as granzyme B (19
). The agonist and variant viral peptides triggered similar increases in the expression of intracellular granzyme B in CD8+
CL-4 T cells early after stimulation (). In addition, CD8+
CL-4 T cells responding to either the agonist or variant epitope upregulated cell surface CD107a (LAMP-1a), which is mobilized from intracellular granule stores to the cell surface of CD8+
T cells in response to acute TCR engagement and serves as a surrogate for granule exocytosis-dependent cytotoxicity (). These data indicate that both the agonist and variant influenza epitopes are capable of driving the differentiation of CD8+
CL-4 T cells, culminating in the acquisition of effector activity.
FIGURE 5 Agonist and variant epitope-stimulated CD8+ CL-4 T cells differentiate into mature effector CTLs. CD8+ CL-4 T cells were cultured with 10−6 M PR/8 agonist or Japan variant peptide-pulsed splenocytes and analyzed by flow cytometry for the expression (more ...)
Our findings to this point indicate that variant epitope stimulation of CD8+
CL-4 T cells results in activation, proliferation, and differentiation into mature effector CTLs followed by rapid and extensive apoptotic cell death. We next wanted to investigate the possible causes of the enhanced apoptosis of variant ligand-stimulated CL-4 T cells. Members of the TNFR family known as death receptors, including FasL and TNFRI, have been demonstrated to mediate apoptotic signals in lymphocytes (20
). We had previously implicated Fas/FasL-dependent interactions between responding CD8+
T cells and dendritic cells in the control of effector T cell numbers in vivo following high-dose lethal influenza virus infection (21
). To determine whether death receptor members of the TNFR superfamily may be playing a role in the marked apoptosis of CD8+
CL-4 T cells, we examined the surface expression of Fas, FasL, TNFRI, and TNFRII at various time points following antigenic stimulation (). We observed no difference in the level of expression of any of these molecules on CL-4 T cells stimulated with either agonist or variant peptide (, and data not shown). Although the expression levels were similar, it was possible that a differential sensitivity to signaling through these receptors might explain the enhanced apoptosis. Therefore, we examined the sensitivity of agonist and variant epitope-stimulated CL-4 T cells to Fas-induced cell death after incubation with an anti-Fas mAb (Clone Jo2; ). Using annexin V as a marker for apoptotic cells, no difference in the sensitivity of agonist or variant epitope-stimulated Tg T cells to Fas-induced apoptosis was observed (). These data suggest that death receptor/ligand interactions on agonist and variant epitope-stimulated CL-4 T cells may not be involved in the enhanced apoptosis of variant ligand-stimulated CD8+
FIGURE 6 Role of death receptors in the enhanced apoptosis of CD8+ CL-4 T cells responding to variant epitope stimulation. CD8+ CL-4 T cells were stimulated in vitro with 10−6 M PR/8 agonist or Japan variant peptide-pulsed splenocytes. A, At day 1 and (more ...)
It has been reported that several costimulatory members of the TNFR superfamily may be important regulators of Ag-driven CD4+
T cell survival (4
). We surveyed the expression of several of these costimulatory receptors (including CD27, 4-1 BB, and OX40) on CL-4 T cells responding to agonist and variant peptide stimulation. We observed no difference in the expression of the majority of these costimulatory receptors (e.g., CD27, 4-1BB) between the two responding T cell populations (data not shown). However, we found that T cells stimulated with the agonist peptide upregulated expression of OX40 by day 1 post-stimulation and maintained elevated OX40 expression until day 4 of culture (Supplemental Fig. 1
, data not shown). By contrast, OX40 was only marginally upregulated on the majority of variant peptide stimulated CL-4 T cells, and expression was not sustained (Supplemental Fig. 1
). This result raised the possibility that differences in OX40 expression on the agonist and variant peptide-stimulated T cells could account for the difference in cell viability between the two activated T cell populations and extent of T cell apoptosis observed. To further explore this possibility, we asked whether blocking the interaction of OX40 displayed on the agonist epitope stimulated T cells with its ligand (OX40L) would enhance apoptosis of the agonist stimulated T cells. We found that the addition of saturating concentrations of a blocking Ab to OX40L did not increase apoptosis of the responding T cells (data not shown). Likewise, our attempt to enhance survival of variant peptide stimulated CL-4 T cells by signaling through the OX40 molecule on these cells also had no effect on the development of accelerated apoptosis (data not shown).
Because survival of activated T cells has been directly linked to level expression of the anti-apoptotic members of the Bcl-2 gene family, we evaluated the kinetics of expression of two critical anti-apoptotic gene products Bcl-2 and Bcl-xL in agonist and variant peptide-stimulated CL-4 T cells by flow-based analysis. We found that agonist-stimulated CL-4 T cells strongly upregulated Bcl-2 expression beginning at day 2 and maintained this elevated level until day 4 poststimulation (, data not shown). In contrast, variant stimulated CL-4 T cells, which expressed considerably lower levels of OX40, also had significantly reduced levels of Bcl-2 for the duration of culture (). Although both agonist and variant epitope-stimulated CL-4 T cells expressed comparable amounts of Bcl-xL at day 1 poststimulation, only PR8-stimulated cultures maintained expression of this molecule through day 3 poststimulation (). These findings suggest that the inability of variant epitope-stimulated CL-4 T cells to upregulate and maintain Bcl-2/Bcl-xL expression may lead to defective survival signaling resulting in the enhanced apoptosis of these cells.
FIGURE 7 Bcl-2 family member expression in agonist and variant epitope-stimulated CD8+ CL-4 T cells. CD8+ CL-4 T cells were stimulated in vitro with 10−6 M PR/8 agonist or Japan variant viral epitopes. A, Cells were analyzed for intracellular Bcl-2 and (more ...)
The BH3-only proteins are one of the main proapoptotic subgroups in the Bcl-2 family (22
). Because recent evidence has described an important role for the BH3-only protein, Bim, in the death of activated T cells (23
), we sought to determine whether Bim levels were changed in CD8+
CL-4 T cells responding to agonist or variant epitope stimulation. To this end, Thy1.2+
CL-4 T cells (the splenocyte stimulators are Thy1.1+
) were purified from agonist or variant epitope-stimulated cultures at days 1 and 2 poststimulation, whole-cell lysates were prepared and subjected to SDS-PAGE and Western blotting for Bim. Although alternative splicing can give rise to three Bim variant proteins, T cells express predominantly BimEL,
which was the isoform detected in this assay (). At day 1 poststimulation, both agonist and variant epitope-stimulated CL-4 T cells expressed similar levels of Bim (). Although Bim expression remained relatively unchanged in variant-stimulated T cells at day 2 poststimulation, there was a significant reduction in the level of Bim expressed in agonist-stimulated cells at the same time interval (). This decreased level of Bim, in combination with the increased expression of Bcl-2 and Bcl-xL, might provide a selective survival advantage in agonist-stimulated CL-4 T cells.
If anti-apoptotic members of the Bcl-2 family are responsible for the reduced survival of variant epitope-stimulated CL-4 T cells, it should be possible to rescue the rapid apoptosis of these cells by increasing Bcl-2 or Bcl-xL expression. To this end, peptidestimulated CL-4 T cells were transduced with a retrovirus vector expressing Bcl-2 or Bcl-xL and GFP and examined for GFP expression. We would expect that if expression of Bcl-2 or Bcl-xL provided a survival advantage, the proportion of GFP+ cells within the live gate would increase. In CL-4 T cell cultures stimulated with the agonist PR8 epitope and transduced with the control vector, the percentage of GFP+ cells remained constant between days 3 and 4, and there was no significant increase in the proportion of GFP+ cells in cultures transduced with Bcl-2 or Bcl-xL–expressing retrovirus (). In contrast, whereas the percentage of GFP+ cells also remained constant with control vector transduction in variant Japan epitope-stimulated cultures, there was a dramatic increase in the proportion of GFP+ cells between days 3 and 4 after retroviral expression of Bcl-2 and Bcl-xL (). These data support the idea that defective survival of variant epitope-stimulated CL-4 T cells is associated with reduced levels of the anti-apoptotic members of the Bcl-2 family.
FIGURE 8 Retroviral expression of Bcl-2 and Bcl-xL suppresses apoptosis of variant epitope-stimulated CD8+ CL-4 T cells. CD8+ CL-4 T cells were stimulated in vitro with 10−6 M PR/8 agonist or Japan variant peptide. On day 1 and 2, cells were transduced (more ...)